Enzymes for reduced immunological stress

ABSTRACT

Compositions suitable for oral administration to an animal comprising at least one immune stress-reducing enzyme in an amount effective to decrease the level of positive acute phase protein in an animal, increase the level of negative acute phase protein in an animal, and/or improve animal growth performance is provided, as are methods using such compositions. The compositions include animal feed compositions, liquid compositions other than animal feed, and solid compositions other than animal feed.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit under 35 U.S.C. §119(e) ofthe filing date of U.S. provisional application 60/750,339, filed Dec.15, 2006, the entire contents of which are incorporated herein byreference.

FIELD OF THE INVENTION

The present invention provides compositions and methods for reducingimmunological stress and improving animal growth performance. Inparticular, the invention provides compositions comprising enzymes thatare effective to reduce immunological stress or that are effective totreat or prevent infection or that are effective to improve animalgrowth performance. The invention also provides methods using thecompositions.

BACKGROUND

An animal may experience immunological stress for a number of reasons,including exposure to an antigen that is recognized by the animal'simmune system. An antigen may trigger an immune response that is anadaptive immune response or that is an innate immune response. When animmune response is triggered, the animal experiences immunologicalstress as its immune system responds to the perceived threat. Often,immunological stress hampers animal growth performance.

Acute phase proteins (APP) are a group of blood proteins whose bloodconcentration changes when an animal is experiencing stress, such asinfection, inflammation, surgical trauma, or other internal or externalchallenges. See, e.g., Murata et al., Vet. J. 168: 28 (2004). APP arebelieved to play a role in an animal's innate immune response. Forexample, APP may be involved in restoring homeostasis and restrainingmicrobial growth until an acquired immunity is developed.

APP include “negative” proteins whose concentration decreases withstress, and “positive” proteins whose concentration increases withstress. See, e.g., Murata et al., supra. Negative APP include albuminand transferrin. Positive APP include proteins synthesized byhepatocytes upon stimulation by pro-inflammatory cytokines and releasedinto the bloodstream, such as haptoglobin, C-reactive protein, serumamyloid A, ceruloplasmin, fibrinogen, and α-1-acid glycoprotein (AGP).Extra-hepatic production of APP also has been reported for mostmammalian species. Id. Pro-inflammatory cytokines such as interleukin-6(IL-6) and tumor necrosis factor α (TNF-α) are believed to be the majormediators of APP synthesis in the liver. Inflammation, infection ortissue injury triggers cytokine release by defense-oriented cells,thereby inducing APP synthesis. The induction of positive APP also isassociated with a decrease in the synthesis of negative APP. Id.

Methods of quantifying APP have been established, and circulating APPconcentration (e.g., serum levels of APP) has been correlated to theseverity of the animal's condition. Id. Thus, APP concentration can beused as an indicator of an animal's immune stress level.

An animal's immune system may recognize antigens that do not pose a realthreat to the animal's health, such as plant- and animal-derivedingredients in animal feed compositions. These antigens may trigger animmune response, such as an innate immune response, thereby causing theanimal to experience immunological stress. This stress response can beidentified and monitored via serum APP concentration.

Even when the immune-triggering antigen did not pose a real threat tothe animal's health, the stress response can have a detrimental effect.This may be observed as a decrease in feed efficiency, a decrease inweight gain rate or decrease in weight, an increase in susceptibility toinfection, or an increase in body temperature, for example.

The use of antibodies, such as anti-phospholipase A2 antibodies, toreduce gastrointestinal inflammation in animals has been described. See,e.g., U.S. Pat. No. 6,383,485. Feed compositions have been describedthat comprise a hemicellulase capable of degrading β-mannan-containinghemicellulose (e.g., a β-mannanase-type hemicellulase), such asendo-1,4-β-mannanase, or a phospholipase, such as phospholipase A2, forimproved feed efficiency. See, e.g., WO 97/41739, U.S. Pat. No.6,162,473, and U.S. Pat. No. 6,183,739.

Likewise described have been feed compositions comprised of an enzyme,such as PI-PLC, that cleaves a linkage, thereby to effect release of acell-surface protein or carbohydrate, for the treatment or prevention ofdigestive tract infection. See, e.g., WO 01/41785. Walsh et al., J.Anim. Sci. 73: 1074 (1995), discuss feed compositions comprisingglucanase enzymes that cleave a mixed link glucan substrate, such as1,4-β-glucanase which cleaves mixed β-1,3, β-1,4-substrates. In ourtests, however, neither PI-PLC nor 1,4-β-glucanase displayedimmune-stress reducing activity.

There has been no description heretofore of a feed composition comprisedof an enzyme that is other than a fl-mannanase-type hemicellulase or aphospholipase and that is present in an amount effective to reduceimmunological stress.

Accordingly, there is a need for compositions and methodology forreducing immunological stress in animals.

SUMMARY OF THE INVENTION

One embodiment provides a composition suitable for oral administrationto an animal comprising an immune stress-reducing enzyme in an orallyacceptable carrier. The composition is selected from the groupconsisting of: (i) an animal feed comprising an amount of the enzymeeffective to decrease the level of positive acute phase protein in theanimal, increase the level of negative acute phase protein in theanimal, and/or improve animal growth performance; (ii) a liquidcomposition other than an animal feed comprising at least 40,000 IUenzyme/L; and (iii) a solid composition other than an animal feedcomprising at least 40,000 IU enzyme/kg. The enzyme is other than aβ-mannanase-type hemicellulase or phospholipase, and, if the enzymecomprises 1,3-β-glucanase, the composition is selected from the groupconsisting of (i) an animal feed comprising at least 20 IU1,3-β-glucanase/kg feed; (ii) a liquid composition other than an animalfeed comprising at least 155,000 IU 1,3-β-glucanase/L and (iii) a solidcomposition other than an animal feed comprising at least 300,000 IU1,3-β-glucanase/kg.

In one embodiment, the composition is an animal feed comprising at least20 IU enzyme/kg feed. In another embodiment, the composition is a solidcomposition other than an animal feed comprising at least 80,000 IUenzyme/kg, or at least 160,000 IU enzyme/kg.

In one embodiment, the composition is an animal feed that comprises aningredient that induces an immune response in the animal and the enzymecomprises an enzyme that degrades said ingredient. In one embodiment,the ingredient is an antigen displayed by a pathogenic microorganism.

In one embodiment, the enzyme comprises 1,3-β-glucanase. In oneembodiment, the enzyme comprises 1,3-β-glucanase and the composition isselected from the group consisting of (i) an animal feed comprising atleast 30 IU 1,3-β-glucanase/kg feed; (ii) a liquid composition otherthan an animal feed comprising at least 230,000 IU 1,3-β-glucanase/L and(iii) a solid composition other than an animal feed comprising at least450,000 IU 1,3-β-glucanase/kg.

Another embodiment provides a composition suitable for oraladministration to an animal comprising two or more immunestress-reducing enzymes, wherein the composition comprises at least oneimmune stress-reducing enzyme other than 1,4-β-mannanase and1,3-β-glucanase. The composition is selected from the group consistingof: (i) an animal feed comprising an amount of said immunestress-reducing enzymes effective to decrease the level of positiveacute phase protein in said animal, increase the level of negative acutephase protein in said animal, and/or improve animal growth performance;(ii) a liquid composition other than an animal feed comprising at leastone immune stress-reducing enzyme in an amount of at least 40,000 IUenzyme/L; and (iii) a solid composition other than an animal feedcomprising at least one immune stress-reducing enzyme in an amount of atleast 40,000 IU enzyme/kg.

In one embodiment, the composition is an animal feed comprising at leastone immune stress-reducing enzyme in an amount of at least 20 IUenzyme/kg feed. In another embodiment, the composition is a solidcomposition other than an animal feed comprising at least one immunestress-reducing enzyme in an amount of at least 80,000 IU enzyme/kg, orat least 160,000 IU enzyme/kg.

In specific embodiments, the composition is selected from the groupconsisting of (i) a composition comprising 1,4-β-mannanase andchitanase; (ii) a composition comprising 1,4-β-mannanase andxyloglucanase; (iii) a composition comprising 1,4-β-mannanase andarabinanase; (iv) a composition comprising 1,3-β-glucanase andchitanase; (v) a composition comprising 1,3-β-glucanase andxyloglucanase; (vi) a composition comprising 1,3-β-glucanase andarabinanase and (vii) a composition comprising 1,4-β-mannanase,1,3-β-glucanase and arabinanase.

Another embodiment provides a composition suitable for oraladministration to an animal comprising 1,4-β-mannanase and1,3-β-glucanase. The composition is selected from the group consistingof (i) an animal feed comprising 1,4-β-mannanase and at least 20 IU1,3-β-glucanase/kg feed, (ii) a liquid composition other than an animalfeed comprising 1,4-β-mannanase and at least 155,000 IU1,3-β-glucanase/L and (iii) a solid composition other than an animalfeed comprising 1,4-β-mannanase and at least 300,000 IU1,3-β-glucanase/kg. In one embodiment, the composition is selected fromthe group consisting of (i) an animal feed comprising 1,4-β-mannanaseand at least 30 IU 1,3-β-glucanase/kg feed; (ii) a liquid compositionother than an animal feed comprising 1,4-β-mannanase and at least230,000 IU 1,3-β-glucanase/L and (iii) a solid composition other than ananimal feed comprising 1,4-β-mannanase and at least 450,000 IU1,3-β-glucanase/kg. In one embodiment, the composition further comprisesone or more additional immune stress-reducing enzymes.

Another embodiment provides a method of improving animal growthperformance and/or reducing immune stress in an animal, comprisingorally administering to the animal any of the compositions describedabove.

In one embodiment, the animal is administered an ingredient that inducesan immune response in the animal and the composition comprises at leastone immune stress-reducing enzyme that degrades the ingredient. In oneembodiment, the ingredient and enzyme are administered in the samecomposition. In one embodiment, the composition is an animal feed. Inone embodiment, the ingredient is an antigen displayed by a pathogenicmicroorganism.

Another embodiment provides a method of preventing or treating infectionassociated with a pathogenic microorganism that displays an antigen,comprising orally administering to an animal in need thereof any of thecompositions described above, wherein the composition comprises at leastone immune stress-reducing enzyme that degrades the antigen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the best curve fit (and underlying polynomial equation) forcalculating the concentration of chicken α-1-acid glycoprotein (AGP) inplasma samples of test chickens with data obtained in Example 1.

FIG. 2 is a Box and Wisker plot graphically showing the AGP levels inchicken serum from test chickens, as described in Example 2. The rangeof the data is represented by the vertical lines. The box represents therange of the data within one standard deviation of the mean. Thehorizontal line indicates the data mean.

FIG. 3 shows the AGP levels of serum from chickens receiving one ofseveral different feeds, including feeds in accordance with theinvention and prior art feeds, as described in Example 3.

FIG. 4 shows the best curve fit (and underlying polynomial equation) forcalculating the concentration of AGP in plasma samples of test turkeyswith data obtained in Example 8.

DETAILED DESCRIPTION

As used in the following discussion, the terms “a” or “an” should beunderstood to encompass one or more, unless otherwise specified.

As used herein, the term “animal” refers to any animal, including humansand other animals, including companion animals such as dogs and cats,livestock, such as cows and other ruminants, buffalo, horses, pigs,sheep, fowl (e.g., chicken, ducks, turkeys, and geese) and aquacultureanimals (e.g., fish and shrimp and eels).

In the present description, the phrases “enzyme that degrades anantigen” and “enzyme that degrades an ingredient” mean that the enzymeconverts the antigen or ingredient to a form that is not recognized bythe animal's immune system. The ability of an enzyme to degrade anantigen or ingredient can be identified by measuring the animal's serumAPP concentration, whereby a decrease in the serum concentration ofpositive APP, or an increase in the serum concentration of negative APP,indicates that the enzyme has degraded the antigen or ingredient.

As noted above, the term “APP” include “negative” proteins whoseconcentration decreases with stress, and “positive” proteins whoseconcentration increases with stress. The invention includes compositionsand methods that increase the concentration of negative acute phaseproteins whose concentration typically decreases with stress, as well ascompositions and methods that decrease the concentration of positiveacute phase proteins whose concentrations typically increase withstress. For convenience, in the discussion that follows, the inventionis exemplified with reference to the effect of the compositions andmethods on positive acute phase proteins. Thus, the term “APP” in thediscussion that follows generally refers to any one or more positiveacute phase proteins associated with an animal's stress response. Itshould be understood that the compositions and methods described hereinas decreasing the concentration of “APP” (referring to positive acutephase proteins) also are useful for increasing the concentration ofnegative acute phase proteins.

One aspect of the invention relates to a composition comprising anenzyme that is effective to reduce the immunological stress experiencedby an animal. For convenience, these enzymes are referred to herein as“immune stress-reducing” enzymes. As used herein, the term “immunestress-reducing enzyme” means any enzyme that degrades an antigen ormolecular pattern that is recognized by the animal's immune system,e.g., an antigen or molecular pattern that triggers an immune response,thereby causing the animal to experience immunological stress. The term“molecular pattern” as used herein includes general molecular patternsthat are bound by receptors in the context of the innate immune system,such as molecular patterns that are usually associated with pathogens.

In accordance with one embodiment, the immune stress-reducing enzyme isnot a β-mannanase-type hemicellulase. In one accordance with thatembodiment, the immune stress-reducing enzyme is notendo-1,4-β-D-mannanase. In accordance with another embodiment, theenzyme is not a phospholipase. In accordance with another embodiment,the immune stress-reducing enzyme is not 1,4-β-D-glucanase. Inaccordance with another embodiment, the immune stress-reducing enzyme isnot PI-PLC. In accordance with another embodiment, the immunestress-reducing enzyme is not a β-mannanase-type hemicellulase or aphospholipase. In accordance with yet another embodiment, the immunestress-reducing enzyme is not a β-mannanase-type hemicellulase, is not1,4-β-glucanase, and is not a phospholipase. In accordance with afurther embodiment, the immune stress-reducing enzyme is not aβ-mannanase-type hemicellulase, is not 1,4-β-glucanase, is not aphospholipase, and is not PI-PLC.

While not wanting to be bound by any theory, the present inventorsbelieve that the immune stress-reducing enzyme's degradation of theantigen or molecular pattern inhibits or reduces the immune responsetriggered by the antigen or molecular pattern, thereby reducing theanimal's immunological stress. The reduction in immunological stress canbe identified and monitored by measuring the animal's serum APPconcentration, using methods known in the art for quantifying APP.Examples of such methods are referenced in Murata, et al., supra, andare described and referenced in Hulten et al., Vet. Microbiol. 95: 75(2003) and Holt et al., supra, as well as in Example 1 below.

In a related embodiment, the invention provides methods for reducingimmunological stress in an animal that comprise administering to theanimal a composition comprising an amount of an immune stress-reducingenzyme effective to reduce the level of APP in the animal.

A number of different positive acute phase proteins have beenidentified, including α-1-acid glycoprotein (AGP), ceruloplasmin (Cp),proteins of the collectin family (e.g., lung surfactant proteins,conglutinin and mannan-binding lectin), fibrinogen (Fb), C-reactiveprotein (CRP), haptoglobin, protease inhibitors (e.g., α-1-antitrypsin,α-1-antichymotrypsin, and α-2-macroglobulin) and serum amyloid-A (SAA).Other potential APP include lipopolysaccharide-binding protein (LPB),phospholipid-binding proteins such as annexins and Major Acute PhaseProtein (MAP). Murata, et al., supra. Serum concentrations of any one ofthese or other APP can be used to identify, assess and monitor enzymeactivity in accordance with the invention.

Different APP may play more significant roles in the stress responses ofdifferent animals. For example, AGP is known to be clinically importantin cattle, and is associated with infection in pigs, dogs, cats andchicken (including hens). Cp has been reported to be an indicator ofinfection in cattle, horses, and chickens. CRP has been identified inruminants, horses, pigs, dogs, and cats, although it has not beendemonstrated that CRP is an APP in cattle. CRP has been shown to beassociated with infection in horses and pigs. Fb is a reliable indicatorof inflammation, bacterial infection or surgical trauma in cattle andsheep, and is associated with infection in horses. Hp is an APP in anumber of production and companion animals, including ruminants such ascattle, sheep, pigs, horses, and dogs. SAA has been associated withinflammation and infection in cattle and with infection in horses, pigs,companion animals such as dogs, and chicken. An increase in SAA milklevels has been found in cows and ewes with mastitis. Serum LBP has beenassociated with infection in cattle, as has local levels of annexins (onthe surfaces of secretory epithelia in lungs of infected cattle). MAP isreported to be an indicator of infection in pigs. Additionally, whiletransferrin is usually considered a negative acute phase protein, itappears to play a role as a positive acute phase protein in chickens.Murata, et al., supra; Holt et al., Poultry Sci. 81: 1295-1300 (2002).Others also have reported that SAA and Hp, as well as CRP and MAP, areassociated with infection in pigs. Hulten et al., supra.

In some embodiments, the compositions of the invention comprise anamount of immune stress-reducing enzyme that is effective to decreasethe serum concentration of APP in an animal. The amount may varydepending on the animal and the immune stress-reducing enzyme, and canreadily be determined by those skilled in the art using methods known inthe art. For example, an animal's serum APP levels can be measured priorand subsequent to administration of the enzyme, or serum APP levels ofequivalent treated and control animals can be compared. (In this regard,it may be advantageous to compare treated and control animals of thesame age, as APP levels may change with age. For example, we have foundthat serum AGP levels increase with chicken age.) A decrease in serumAPP concentration associated with administration of the enzyme indicatesthat an effective amount of enzyme was administered.

In other embodiments, the compositions of the invention comprise anamount of immune stress-reducing enzyme that is effective to improveanimal growth performance (also referred to as “live performance,”particularly in the field of poultry). As used herein, the phrase“animal growth performance” includes any parameter that reflects animalgrowth, including feed conversion, water absorption, feces watercontent, uniformity of body weight within a flock or group of animals,livability, and mortality. While not wanting to be bound by any theory,it is believed that, under some conditions, the effect of the immunestress-reducing enzyme on APP concentration is masked by factors such asimmune stress-inducing factors, such as the presence of a low-levelinfection in a group of animals or stressful living conditions. Undersuch conditions, the immune stress-reducing enzyme may nevertheless beeffective to improve animal growth performance. Thus, animal growthperformance is an alternative measure of the effectiveness of thecompositions and methods of the present invention.

The composition may be any composition suitable for administration to ananimal. In one embodiment, the composition is suitable for oraladministration. In one specific embodiment, the composition that issuitable for oral administration is generally recognized as safe fororal administration to an animal. In another specific embodiment, thecomposition that is suitable for oral administration contains onlyingredients, and amounts of said ingredients, that are generallyrecognized as safe for oral administration to an animal. In anotherspecific embodiment, the composition that is suitable for oraladministration does not contain any ingredients, or amounts of saidingredients, that are not generally recognized as safe for oraladministration to an animal. In another specific embodiment, thecomposition that is suitable for oral administration contains onlyingredients, and amounts of said ingredients, that are allowed, or thatare not prohibited, for oral administration to an animal. In anotherspecific embodiment, the composition that is suitable for oraladministration does not contain any ingredients, or amounts of saidingredients, that are not allowed, or that are prohibited, for oraladministration to an animal.

In some embodiments, the composition comprises an orally acceptablecarrier for the enzyme. As used herein, “orally acceptable carrier”includes any physiologically acceptable carrier suitable for oraladministration. Orally acceptable carriers include without limitationanimal feed compositions, aqueous compositions, and liquid and solidcompositions suitable for use in animal feed products and/or for oraladministration to an animal. Suitable carriers are known in the art, andinclude those described in U.S. Pat. No. 6,780,628.

In some embodiments, the composition is an animal feed. As used herein,the term “animal feed” has its conventional meaning in the field ofanimal husbandry. For example, animal feed includes edible materialswhich are consumed by livestock for their nutritional value. Animal feedincludes feed rations, e.g., compositions that meet an animal'snutritional requirements, and also include compositions that do not meetan animal's nutritional requirements.

In specific examples of such an embodiment, the amount of enzyme is atleast about 50,000 international units (IU) per U.S. ton of feed, atleast about 60,000 IU per ton of feed, at least about 70,000 IU per tonof feed, at least about 80,000 IU per ton of feed, at least about 90,000IU per ton of feed, at least about 100,000 IU per ton of feed, at leastabout 200,000 IU per ton of feed, or at least about 500,000 IU per tonof feed, or higher.

In other specific examples, the invention provides an animal feedcomprising an amount of immune stress-reducing enzyme of at least about20 IU/kg feed, such as at least 20 IU/kg feed, at least at 25 IU/kgfeed, at least at 30 IU/kg feed, at least at 35 IU/kg feed, at least at40 IU/kg feed, at least at 45 IU/kg feed, at least 50 IU/kg feed, ormore. While not wanting to be bound by any theory, it is believed thatan animal feed comprising an amount of immune stress-reducing enzyme ofat least about 20 IU/kg feed will be effective to decrease the level ofpositive acute phase protein in said animal, increase the level ofnegative acute phase protein in said animal, and/or improve animalgrowth performance.

Thus, in some embodiments, the invention provides an animal feedcomprising an amount of immune stress-reducing enzyme effective todecrease the level of positive acute phase protein in the animal,increase the level of negative acute phase protein in the animal, and/orimprove animal growth performance

The feed composition may be prepared by methods known in the art. Forexample, immune stress-reducing enzyme can be added to the other feedingredients at any stage during the manufacturing process, as deemed tobe appropriate by those skilled in the art. In one embodiment, theenzyme is provided as a solution, such as a liquid enzyme concentratethat is added to other feed ingredients during the manufacturingprocess. Alternatively, an enzyme-containing solution is sprayed on to asubstantially final form of the animal feed. In another embodiment, theenzyme is provided as a solid composition (such as a powder), such as asolid composition that is added to other feed ingredients during themanufacturing process. Exemplary methods for manufacturingenzyme-containing feed are described in WO 97/41739.

In some embodiments, the composition is other than an animal feed. Forexample, the composition may be a liquid composition other than ananimal feed or a solid composition other than an animal feed. Suchcompositions may be suitable for direct administration to an animal ormay be used as a feed additive (e.g., added to feed prior to feeding) ora feed supplement (including supplements that are diluted with otherfeed components prior to feeding and supplements that are offered to ananimal on a free choice, separate basis). Examples of a liquidcomposition other than an animal feed include liquid enzymeconcentrates, including liquid enzyme concentrates that are typicallydiluted or combined with other ingredients prior to oral administrationto an animal.

In embodiments where the composition is a liquid composition other thanan animal feed, such as an enzyme solution, the liquid composition orsolution may comprise at least about 40,000 international units (IU) perliter of solution, such as at least 40,000 IU/L, at least 50,000 IU/L,at least 60,000 IU/L, at least 70,000 IU/L, at least 80,000 IU/L, atleast 90,000 IU/L, at least 100,000 IU/L, at least about 500,000 IU/L,at least about 600,000 IU/L, at least about 700,000 IU/L, at least about800,000 IU/L, at least about 900,000 IU/L, at least about 1,000,000IU/L, at least about 2,000,000 IU/L, or at least about 5,000,000 IU/L.

In some embodiments, an amount of liquid composition other than ananimal feed, such as about 500 mL solution, is applied to or combinedwith an amount of feed, such as to a ton of feed, to arrive at feedformulations with enzyme levels described above. In other embodiments,an amount of liquid composition other than an animal feed is applied toor combined with an amount of feed to prepare an animal feed with anamount of enzyme effective to decrease the level of positive acute phaseprotein in the animal, increase the level of negative acute phaseprotein in the animal, and/or improve animal growth performance.

It is believed that currently available liquid enzyme concentratecompositions (other than the 1,3-β-glucanase compositions discussedbelow) that are suitable for oral administration comprise much less thanat least about 40,000 IU/L of an immune stress-reducing enzyme, if anyat all, and are not effective to decrease the level of positive acutephase protein, increase the level of negative acute phase protein,and/or improve animal growth performance, when used in accordance withtheir instructions.

In embodiments where the composition is a solid composition other thanan animal feed, the composition may comprise at least about 40,000IU//kg, such as at least 40,000 IU/kg, at least 50,000 IU/kg, at least60,000 IU/kg, at least 70,000 IU/kg, at least 80,000 IU/kg, at least90,000 IU/kg, at least 100,000 IU/kg, at least 120,000 IU/kg, at least140,000 IU/kg, at least 160,000 IU/kg, at least 180,000 IU/kg, at least200,000 IU/kg, or more.

In some embodiments, an amount of a solid composition other than ananimal feed is applied to or combined with an amount of feed to arriveat feed formulations with enzyme levels described above. In otherembodiments, an amount of solid composition other than an animal feed iscombined with an amount of feed to prepare an animal feed with an amountof enzyme effective to decrease the level of positive acute phaseprotein in the animal, increase the level of negative acute phaseprotein in the animal, and/or improve animal growth performance.

It is believed that currently available solid enzyme powder compositionsthat are suitable for oral administration comprise much less than atleast about 40,000 IU/kg of an immune stress-reducing enzyme, if any atall, and are not effective to decrease the level of positive acute phaseprotein, increase the level of negative acute phase protein, and/orimprove animal growth performance, when used in accordance with theirinstructions.

As conventional in the art, the term “IU” or “international unit” refersto an amount of enzyme that will catalyse the transformation of 1micromole of the substrate per minute under conditions that are optimalfor the enzyme. Weight equivalents to international units of immunestress-reducing enzymes are known in the art and can be determined usingstandard assays. Exemplary standard assays for representative immunestress-reducing enzymes are outlined below.

In one embodiment, the enzyme is expressed by a plant that is used inanimal feed. For example, corn can be genetically engineered to expressan immune stress-reducing enzyme and the resulting genetically modifiedcorn product can be used in feed. Production also can be effected withother genetically modified or classically modified systems such asbacteria, e.g., E. coli, Bacillus sp., Lactobacillus; yeast, e.g.,Pichia, Yarrow, Saccharomyces, Schizosaccharomyces (e.g.,Schizosaccharomyces pomb, Hansenula, Kluyveromyces, Candida), and otherfungus, such as Aspergillus, Rhizopus, Tricoderma, Humicola,Penicillium, and Humicola.

In accordance with another embodiment, the immune stress-reducing enzymeis provided in a capsule or tablet from for oral ingestion. Theinvention also encompasses embodiments where the enzyme is administeredby other routes, such as intravenously, peritoneally, or subcutaneously,as a component of a composition formulated for such administration inaccordance with known pharmacological practices.

An animal's immune system may recognize as an antigen or molecularpattern certain ingredients of a feed composition that do not pose areal threat to the animal's health. Nonetheless, the ingredient triggersan immune response that causes the animal to experience immunologicalstress, and that can be identified and monitored by an increase in theserum concentration of one or more APP. While not wanting to be bound byany theory, the present inventors believe that this “unnecessary andcounterproductive” immune response may involve pattern recognitionreceptors (PRR), such as those involved in the innate immune system.

The innate immune system provides an immune response that does notdepend on specific antigen recognition. See, e.g., Tosi, J. AllergyClin. Immunol. 116: 241 (2005). One aspect of the innate immune systeminvolves PRR, which recognize and bind pathogen-associated molecularpatterns, transducing immune response signals. See, e.g., Fabrick etal., J. Biol. Chem. 279: 26605 (2004). Examples of PRR include Toll-likereceptors (TLR) that recognize a range of molecular patterns andgenerate intracellular signals for activation of a range of hostresponses. See, e.g., Tosi, supra; Blach-Olszewska, Arch. Immunol. Ther.Exp. 53: 245 (2005). PRR/TLR have been identified that recognize mannose(e.g., Blach-Olszewska, supra), 1,3-β-glucan (e.g., Rice et al., J.Leukoc. Biol. 72:140 (2002)), lipopolysaccharide and phosphorylcholine(e.g., Baumgarth et al., Semin. Immunopathol. 26: 347 (2005)),lipoteichoic acid, phenol-soluble modulin, muramyl dipeptide andpeptidoglycan (e.g., Fournier et al., Clin. Microbiol. Rev. 18: 521(2005). Immunomodulatory receptors for mannan (e.g., Klabunde et al.,Parasitol. Res. 88: 113 (2002) (mannan-binding lectin)), andN-acetyl-D-glucosamine and N-acetyl-D-mannosamine (e.g., Hansen et al.,J. Immunol. 169: 5726 (2002)). TLRs for double stranded RNA (e.g., Bellet al., Proc. Nat'l Acad. Sci. USA 102: 10976 (2005)) and DNA withmethylation patterns that differ from endogenous DNA (e.g., Huang atal., J. Immunol. 175: 3964 (2005); Nonnemacher et al., Infect. Immun.71: 850 (2003)) also have been identified.

While these molecular patterns are associated with pathogenicmicroorganisms (e.g., bacteria, viruses, fungi and protozoa) they alsoare presented by some non-pathogenic molecules, such as animal feedingredients. An innate immune response to non-pathogenic moleculespresenting these molecular patterns unnecessarily subjects an animal toimmunological stress, and may detrimentally impact the animal's feedefficiency, slow the animal's rate of weight gain or result in weightloss, make the animal more susceptible to infection, increase theanimal's body temperature, or otherwise have a negative impact on theanimal's health or food energy (calorie) utilization efficiency. Theinnate immune response resulting from MBL (mannose-binding lectin)function, for example, induces powerful responses. It has been shownthat mutation of one of the mannose binding protein's genes in miceparadoxically allows survival from a normally lethal acute septicperitonitis challenge (Takahashi, K. et al., Microbes Infect. 4 (8):773-784, 2002). The immune stress from aggressive innate immune responseis more lethal than the infection in this case.

β-mannan is a component of soybean products and soybean-based animalfeeds. High molecular weight forms of β-mannan present in animal feedcan trigger an “unnecessary and counterproductive” innate immuneresponse, thereby subjecting the animal to immunological stress. Thepresent inventors found that this immunological stress can be reduced orprevented using a β-mannanase-type hemicellulase,endo-1,4-β-D-mannanase, an enzyme which degrades β-mannans (e.g.β-galactomannan, β-glucomannan), thereby reducing or preventing theimmune response to β-mannan. As shown in the Examples below, thereduction in immunological stress is reflected in a decrease in serumAPP concentration.

α-mannanase, which degrades α-mannan, is useful as an immune-stressreducing enzyme in accordance with the invention. α-mannan is notconsidered to be a hemicellulose because it does not sharecharacteristic properties of hemicelluloses.

In the field of industrial enzymes, the term “hemicellulase” has beenused as a trade name for β-mannanase. Likewise, patents and publicationsco-authored by the inventors use the term “hemicellulase” to refer toβ-mannanase, including endo-1,4-β-D-mannanase. See, e.g., U.S. Pat. No.6,162,473. In other contexts, the term “hemicellulase” may be broader,encompassing glucanases and xylanases in addition to mannanase, asexplained below.

The term “hemicellulose” was coined to describe carbohydrate plantmaterial obtained by extraction with a dilute alkaline solution that ishydrolyzed more easily than cellulose. See, e.g., Schulze, E., Berichteder Deutschen Botanischen Gesellschaf, 24: 2277 (1891); Schulze, E., Z.Physiol. Chem. 16: 387 (1892). Since then, “hemicellulose” has come tospecify water insoluble plant polysaccharides associated with cellulose,other than pectin and starches and polysaccharides in plant sap, thatare soluble in dilute alkali solutions. See, e.g., Whisler et al.,“Hemicelluloses,” in IV POLYSACCHARIDE CHEMISTRY 112 (Academic Press,1953). Xylan, β-mannans and galactans are generally considered to behemicelluloses, although some β-mannans, like Locust bean gum and guargum galacotmannans are readily soluble. Softwood trees have a lot ofβ-mannans associated with their cellulose and hardwoods have a lot ofxylans.

In contrast to hemicelluloses, α-mannan is associated with fungal cellswalls, such as Saccharomyces, is not a structural component of wood, andis uniformly found in eucaryotic glycoproteins that are generallysoluble in water. Thus, α-mannan is not considered to be ahemicellulose, and α-mannanase is not a hemicellulase. α-mannanase isuseful as an immune stress-reducing enzyme in accordance with thepresent invention because it degrades α-mannans that are recognized byan animal's immune system, but that are not pathogen associated. Theinnate immune system is sensitive to mannan because polymers containingmannose are found on the surface of many pathogens.

Other feed ingredients that may be recognized by an animal's immunesystem include β-1,3-glucan (a common structural component of plantmaterials), N-linked glycoprotein complexes (found, for example, insoybean products), double-stranded RNA from plants, animals or microbes,and DNA from microbes, plants or animals with a foreign (non-endogenous)methylation pattern. Thus, in accordance with one embodiment, theinvention provides a composition comprising one or more immunestress-reducing enzymes that degrade one or more of these or other feedingredients. In a related embodiment, the invention provides methods forreducing immunological stress in an animal that comprise administeringto the animal a composition comprising an effective amount of such anenzyme or enzymes. Specific examples of immune stress-reducing enzymesand the antigens they degrade are set forth in the following table. Theinvention encompasses compositions that comprise other immunestress-reducing enzymes that degrade the same or different antigens, aswell as the use of such other enzymes to reduce immunological stress.

ANTIGENS ENZYMES α-mannan α-mannanase α-mannosidase β-mannansβ-mannanase hemicellulase (β-mannanase type) 1,4-β-mannanaseendo-1,4-β-D-mannanase β-1,3-glucans 1,3-β-glucanaseEndo-1,3-β-glucanase (EC 3.2.1.39) β-glucosidase double stranded RNAnon-specific nuclease non-capped mRNA RNAse L 3pRNA dsRNA specificadenosine deaminase DNA DNAase non-specific nuclease CG specificrestriction endonuclease N-linked glycoproteins carbohydrases (e.g.,asialoglycoprotein) N-glycanases endo enzymes PNGases phosphocholine insphingomyelin sphingomyelinase N-acetlyglucosamine containing chitinasepolymer, (e.g., chitin) (EC 9 3.2.1.14) chitin deacetylase carbohydratedeacetylase N-acetylglucosaminidase phosphatidylserinephosphatidylserine decarboxylase phospholipase C phospholipase Dsulfated galactoside-saccharide sulfatase β-galactoside β-galactosidasexyloglucan xyloglucanase (EC 3.2.1.15) lipoarabinomannan (LAM)arabinanase arabinogalactan (AG) hyaluronan (hyaluronic acid)hyaluronidase (EC 3.2.1.35) arabinogalactan and otherα-arabinofuranosidase arabino-modifided carbohydrates chondroitinsulfate chondroitinase glucocerebrosides glucocerebrosidase methylesters of carbohydrates methyl esterase ferulic acid esterified ferulicacid esterase carbohydrates furuloyl esterase acetylated carbohydratepolymer acetyl esterase carbohydrate deacetylase

In accordance with some embodiments, the invention provides acomposition comprising two or more immune stress-reducing enzymes. Inone embodiment, at least one of the two or more enzymes is not1,4-β-mannanase or 1,3-β-glucanase. In another embodiment, a compositioncomprises 1,4-β-mannanase and 1,3-β-glucanase.

In one specific embodiment, the composition is an animal feed comprising1,4-β-mannanase and at least about 20 IU 1,3-β-glucanase/kg feed, suchas at least 20 IU/kg feed, at least 25 IU/kg feed, at least 30 IU/kgfeed, at least 35 IU/kg feed, at least 40 IU/kg feed, at least 45 IU/kgfeed, at least 50 IU/kg feed, or more, of 1,3-β-glucanase.

In another specific embodiment, the composition is a liquid compositionother than an animal feed comprising 1,4-β-mannanase and at least about155,000 IU 1,3-β-glucanase/L, such as at least 155,000 IU/L, at least230,000 IU/L, at least 300,000 IU/L, at least 380,000 IU/L, or more, of1,3-β-glucanase.

In another specific embodiment, the composition is a solid compositionother than an animal feed comprising 1,4-β-mannanase and at least about300,000 IU 1,3-β-glucanase/kg, such as at least 300,000 IU/kg, at least450,000 IU/kg, at least 600,000 IU/kg, at least 750,000 IU/kg, at least900,000 IU/kg, or more, of 1,3-β-glucanase.

In another embodiment, a composition comprises 1,4-β-mannanase andxyloglucanase. In another embodiment, a composition comprises1,3-β-glucanase and xyloglucanase. In another embodiment, a compositioncomprises 1,4-β-mannanase and chitinase. In another embodiment, acomposition comprises 1,3-β-glucanase and chitanase. In anotherembodiment, a composition comprises 1,4-β-mannanase and arabinanase. Inanother embodiment, a composition comprises 1,3-β-glucanase andarabinanase. In another embodiment, a composition comprises1,4-β-mannanase, 1,3-β-glucanase and arabinanase.

It will be understood that these combinations are exemplary only, andthe invention includes compositions comprising other combinations ofimmune stress-reducing enzymes. For example, the invention includescompositions comprising any one or more of the immune stress-reducingenzymes listed above and/or discussed below and 1,4-β-mannanase.

Immune stress caused by a feed ingredient may not always be an innateimmune system response. It is well known that a certain small percentageof infants fed soy protein-based human milk-replacer formula develop astrong detrimental immunological-based intestinal reaction (see thereport from the Committee on nutrition, American Academy of Pediatrics,Pediatrics 101 (1): p 148, (1998)). N-linked Gycoproteins in soy, forexample β-conglycinin, some times referred to as 7S globulin (Ogawa T,et al., Biosci. Biotechnol. Biochem. 59(5):831-833, 1995; Burks A W, etal., J Pediatr. Gastroenterol. Nutr. 8(2):195-203, 1989) can be strongantigens and are recognized as having anti-nutritional qualities.β-conglycinin is deliberately removed from soy protein isolatepreparations used for nutritional supplements despite its contributionto total protein. Hydrolysis destroys the antigenicity. In addition, wehave found that an enriched 7S soy glycoprotein fraction used in feedingroosters was less well digested than another less glycosylated soyprotein fraction.

Examples of suitable enzymes for degrading carbohydrates in N-linkedglycoproteins include α-fucosidases such as α-1,2-fucosidase andα-1,3-1,4-fucosidase, α-mannosidases such as α-1,6-mannosidase,α-1,2-mannosidase, and α-1,3-mannosidase, β-1,4-galactosidase,endo-β-N-acetylglucosaminidase F (endo F),peptide-N—(N-acetyl-beta-glucosaminyl)asparagine amidase F (PNGase F),PNGase A, endo-β-N-acetylglucosaminidase H (endoH), endo D, endo C,α-N-acetylgalactosaminidase, β-1,3-galactosidase,endo-N-acyl-neuraminidase (endo N), α-2,3-neuraminidase,α-2,6-neuraminidase, α-2,8-neuraminidase, β-N-acetylhexosaminidase,endo-β-N-galactosidase, endo-α-N-acetylglactosaminidase,endo-α-1,6-D-mannanase, arabinogalactanase, α-galactosidase,β-galactosidase.

These enzymes are known in the art and some are available fromcommercial sources. Alternatively, immune stress-reducing enzymes can beobtained from microorganisms that produce enzymes, such as bacteria,fungi and yeast. Additionally, the enzymes can be obtained usingrecombinant technology methods known in the art, by, for example,genetically engineering a host cell to produce an enzyme, e.g., causingtranscription and translations of a gene encoding the enzyme. The aminoacid sequences of a number of the enzymes set forth above are known inthe art. Using those sequences or known nucleotide sequences encodingthose sequences, those skilled in the art can design suitable genes forrecombinant expression of the enzymes. Additionally or alternatively, anucleotide sequence encoding a known immune stress-reducing enzyme canbe used to probe a DNA library to identify other nucleotide sequencesencoding immune stress-reducing enzymes. As known in the art, such a DNAlibrary can be derived from a defined organism or population oforganisms, or can be obtained from natural sources and thus representDNA from microorganisms that are difficult to culture.

In embodiments where the composition comprises a combination of enzymes,the enzyme may be produced individually, by separate organisms, or twoor more of the enzymes may be produced by a single organism. Forexample, a single organism can be recombinantly engineered to producetwo or more enzymes by methods known in the art.

As discussed above, an animal's immune system recognizes a number ofdifferent molecular patterns displayed by pathogenic microorganisms,including lipopolysaccharide (associated with, for example, gramnegative bacteria), bacterial flagella containing the conserved proteinflagellin, peptidoglycan (associated with, for example, gram positivebacteria), lipotechoic acid (associated with, for example, gram positivebacteria) is bound by the C-type lectin L-Ficolin, (Lynch, N.J., et al.,J. Immunology 172: 1198-1202, 2004), phosphorylcholine (associated with,for example, gram positive and gram negative bacteria), DNA (such asbacterial DNA with CpG non-methylated motifs, see Van Uden and Raz, JAllergy Clin Immunol. 104(5):902-10, 1999.), and double-stranded RNA and3pRNA (Hornung, et. al., Science 314: 994-997, 2006). The immuneresponse to these molecules includes an increase in serum APP.

Other pathogenic molecular patterns include N-acetylglucosaminecontaining molecules and N-acetylmannosamine containing molecules. Theexact binding specificity of all collectins (mannose-binding lectin is acollectin or C-type lectin) may not be known, but binding to a number ofdifferent bacterial pathogens is observed by for example, H-ficolin,surfactant-associated protein A (SP-A), and conglutinin. Compounds likeN-acetylglucosamine and N-acetylmannosamine can inhibit the binding andthus are presumed to be part of the pattern recognition bindingspecificity (Haurum, J. S., et al., Biochemical J. 293 (3): 873-878,1993).

Examples of other antigens and molecular patterns that can be targetedfor enzyme degradation in accordance with the invention includebacterial lipoproteins (Hacker, H. et al., J. Exper. Med. 192 (4):595-600, 2000); β-1,3-glucan binding by the collectin Dectin-1 (Adachi,Y., et al., Infection and Immunity 72 (7): 4159-4171, 2004); flagellin(which bind the TLF5) (Honko, A. N., and Mizel, S. B., Immunol. Res. 33(1): 83-101, 2005); fucosyl glycoconjugates; α-Gal-ceramide; fibrinogen;heparin sulfate; sulfated gal-saccharide; chitosan, N-acetylglucosamine;asialoglycoprotein; and β-galactosides.

The class of receptors called scavenger receptors (SR) are structurallyrelated to some of the innate immune response receptors, and may createimmune stress. It is believed that SR are involved in the recycle andclean up of apoptosis or otherwise damaged cells. The scavengerreceptors (SR) expressed by macrophages and dendritic cells are alsoreceptors for the innate immune system. Moreover, some SR recognizepathogens and some innate immune receptors are shown to be important forapoptosis. Thus, in accordance with one embodiment of the invention, themolecular pattern binding targets of SR are targeted for enzymedegradation.

One such SR molecular pattern-binding target is oxidized low densitylipoprotein (LDL). The receptors called LOX-1 (Peiser, L., et al.,Current Opinion in Immunology 14:123-128, 2002) SR-PSOX/CXCL-16(Fukumoto, N., et al., J. Immunol. 173(3): 1620-1627, 2004) and CD36(Bruni, F., et al., Clin. Appl. Thromb. Hemost. 119(4): 417-28, 2005)bind oxidized-LDL that may be present in some feeds, particularly feedscontaining animal by-product meals such as blood meals.

Another SR molecular pattern binding target is phosphatidlyserine (PS)and lyso phosphatidlyserine (lyso PS). SR for PS include SR-PSOX/CXCL-16and other PS receptors (Schlegel, R. A. and Williamson, P., Cell DeathDiffer. 8 (6): 545-548, 2001). Exposure to phosphatidylserinephospholipids may lead to inflammatory responses and phosphatidylserinephospholipids are believed to present in most feeds at some level.

Hyaluronan is abundant in extracellular fluids in animals, but is alsorecognized by innate immune/scavenger system mechanisms, for example, inwound healing. See, e.g., Jameson, et al., J. Expt. Medicine 210 (8):1269-1279, 2005. Chicken combs are a commercial source of hyaluronan,typically used in the purified form of hyaluronic acid. Thus, poultrymeal made from byproducts of meat processing can contain hyaluronan,often in abundant amounts. Hyaluronidase (EC 3.2.1.35), which degradeshyaluronan and hyaluronic acid, is useful as an immune-stress reducingenzyme in accordance with the invention, particularly in the context ofanimals that are fed poultry meal. For example, hyaluronidase is usefulin reducing immune stress associated with feeding poultry meal.

Enzymes that degrade any of these molecular patterns thereby inhibit orreduce the immune response, thus reducing the animal's immunologicalstress. For example, DNAases and non-specific nucleases are known thatdegrade double-stranded RNA and bacterial DNA. Restriction endonucleaseenzymes specific for methylated CG motifs in non-mamalian DNA are known.Enzymes that degrade phosphorylcholine include phosphorylcholinehydrolyase, alkaline phosphatase, acid phosphatase, phosphorylcholineesterase, and phosphorylcholine phosphatase.

This stress reduction can be identified and monitored by measuring thelevel of serum APP, as described above, with decreased serum APPconcentrations reflecting reduced immunological stress.

As noted above, the composition comprises an amount of immunestress-reducing enzyme that is effective to decrease the level of acutephase protein in the animal. This amount may vary from animal to animal,and from enzyme to enzyme, but readily can be determined by thoseskilled in the art, for example, by measuring APP levels, as describedabove. For example, an animal's serum APP levels can be measured priorand subsequent to administration of the enzyme, or serum APP levels oftreated and control animals can be compared. In embodiments where theeffective amount is assessed by measuring serum APP levels prior andsubsequent to administration of the enzyme, the subsequent measurementcan be made from at least about one day to at least about several daysor longer after initial administration of the enzyme. A decrease inserum APP concentration associated with administration of the enzymeindicates that an effective amount of enzyme was administered. It shouldbe understood, however, that APP levels generally decrease as theanimal's adaptive immune response takes effect.

In accordance with some embodiments, the present invention providescomposition comprising 1,3-β-glucanase in an amount effective toeffective to decrease the level of positive acute phase protein in saidanimal, increase the level of negative acute phase protein in saidanimal, and/or improve animal growth performance. In one specificembodiment, the composition is an animal feed comprising at least about20 IU 1,3-β-glucanase/kg feed, such as at least 20 IU/kg feed, at least25 IU/kg feed, at least 30 IU/kg feed, at least 35 IU/kg feed, at least40 IU/kg feed, at least 45 IU/kg feed, at least 50 IU/kg feed, or more,of 1,3-β-glucanase. In another specific embodiment, the composition is aliquid composition other than an animal feed comprising at least about155,000 IU 1,3-β-glucanase/L, such as at least 155,000 IU/L, at least230,000 IU/L, at least 300,000 IU/L, at least 380,000 IU/L, or more, of1,3-β-glucanase. In another specific embodiment, the composition is asolid composition other than an animal feed comprising at least about300,000 IU glucanase/kg, such as at least 300,000 IU/kg, at least450,000 IU/kg, at least 600,000 IU/kg, at least 750,000 IU/kg, at least900,000 IU/kg, or more, of 1,3-β-glucanase.

In some animal feed embodiments where the enzyme comprises1,3-β-glucanase, the enzyme may be present in amount that is at leastabout 100,000 IU per ton feed.

These amounts are much higher than the 1,3-β-glucanase content ofcommercial feed enzyme additives and commercially available feeds, whichthe present inventors have analyzed and found to provide at most fromabout 10,000 IU/ton feed, about 72,500 IU/L non-feed liquid composition,or about 150,000 IU/kg non-feed solid composition. The present inventorsdo not believe that 10,000 IU/ton feed 1,3-β-glucanase would beeffective to reduce APP, and confirmed this belief experimentally. Thepresent inventors also determined experimentally that commercialproducts such as Avizyme® (Danisco A/S, Langebrogade 1, Dk-1001,Copenhagen, Denmark) and Rovobio (Adisseo France SAS, 42, AvenueAristide Briand, BP100, 92164 Antony Cedex,) and commercial feedscomprising standard amounts of β-1,3-1,4-glucanase (Brewzyme™ BG plus,Dyadic International, 140 Intracoastal Pointe Drive, Suite 404, Jupiter,Fla. 33477-5094), xylanase (Multifect® XL, Genencor International, Inc.,925 Page Mill Road, Palo Alto, Calif.), PI-PLC (ChemGen Corp., 211 PerryParkway, Gaithersburg, Md.) and amylase (Amylase FRED, GenencorInternational, Inc., 925 Page Mill Road, Palo Alto, Calif.) do notreduce APP. See Example 3 below and FIG. 3. In the cases where1,3-β-glucanase activity is present, it is within the low ranges notedabove and not effective to reduce AGP.

In another embodiment, the immune stress-reducing enzyme is provided asa component of a composition that also comprises the antigen ormolecular pattern containing compounds that are degraded by the enzyme.For example, the invention includes an animal feed comprisingβ-1,3-glucan and a 1,3-β-glucanase; an animal feed comprising DNA ordouble-stranded RNA and a DNAase or and non-specific nucleases; ananimal feed comprising an N-linked glycoprotein and an endo- orexo-carbohydrase, N-glycanase, or PNGase, or any of the other enzymesset forth above. Other suitable combinations of antigens and immunestress-reducing enzymes will be apparent to those skilled in the art,and are encompassed by the invention.

In this embodiment, it is expected that serum APP levels will remainelevated as long as the composition is administered. Thus, if theeffective amount of immune stress-reducing enzyme is assessed bymeasuring serum APP levels prior and subsequent to administration of theenzyme, the subsequent measurement can be made days or weeks afterinitial administration of the enzyme.

As noted above, the invention includes methods of reducing immune stressin an animal, comprising administering to the animal a compositioncomprising an immune stress-reducing enzyme in an amount effective todecrease the level of acute phase protein in the animal. The compositionmay be any composition described above, including an oral composition,such as animal feed, a liquid composition other than an animal feed, ora solid composition other than an animal feed. The animal may be anyanimal, including a human, and may be a healthy animal or an animalsuffering from infection or other disease or condition.

The invention also includes methods of improving animal growthperformance, comprising administering to the animal a compositioncomprising an immune stress-reducing enzyme. In some embodiments, thecomposition comprises an amount of immune stress-reducing enzymeeffective to improve animal growth performance. The composition may beany composition described above, including an oral composition, ananimal feed, a liquid composition other than an animal feed, or a solidcomposition other than an animal feed. The animal may be any animal,including a human, and may be a healthy animal or an animal sufferingfrom infection or other disease or condition.

In one embodiment, the enzyme is expressed by a plant that is used inanimal feed. For example, corn can be genetically engineered to expressan immune stress-reducing enzyme and the resulting genetically modifiedcorn product can be used in feed.

In one embodiment, the animal is administered the immune stress-reducingenzyme and also is administered the antigen (e.g., thepattern-containing molecule degraded by the enzyme). The enzyme andantigen may be administered separately or simultaneously as part of thesame or different compositions. In one embodiment, the animal isadministered a feed comprising the antigen or pattern containingmolecule, and is separately administered a composition comprising theimmune stress-reducing enzyme. In another embodiment, the animal isadministered a feed comprising the antigen or pattern containingmolecule and a feed supplement comprising the enzyme. In anotherembodiment, the animal is administered a feed comprising both theantigen and the enzyme.

Another aspect of the invention provides compositions and methods forreducing immunological stress by preventing and treating infectioncaused by pathogenic microorganisms. Sometimes animals consumecompositions, such as water or animal feed, that comprise pathogenicmicroorganisms (e.g., bacteria, viruses, fungi and protozoa), or areotherwise exposed to such pathogens. The present invention providescompositions comprising an enzyme that degrades pathogenicmicroorganism's key components (i.e., a “pathogenic component”), in anamount effective to decrease infection and therefore the level of APPexpressed in the animal responding to the infection. The composition isuseful for reducing immunological stress through preventing orminimizing the infection thereby decreasing the immunological stresscaused directly by the pathogen. In one particular aspect of thisembodiment, the invention provides a method preventing and treatingdigestive tract infection.

By degrading pathogen components, enzymes may also treat or preventinfection. That is, because a pathogenic component is degraded, thepathogen could lose its ability to infect the host. This decrease inactual infection would result in reduced immune stress and reduction inserum APP by a different mechanism than described above, but in practiceindistinguishable in terms of the observed APP reduction. There are atleast three scenarios where enzymatic treatment could have a positiveresult. If the pathogen molecular structure degraded by the enzyme isinvolved in binding of the pathogen to the host cells, the first steprequired for infection, or any other key step necessary for successfulinfection, then enzymatic treatment could help. Alternatively, thebinding structure on the host cell might be modified. For example anumber of bacterial and protozoan pathogens have been shown in interactwith proteoglycans on the eukaryotic host cells surface, particularlysulfated proteoglycans (Flekenstein, J. M. et al., Infection andImmunity 70 (3): 1530-1537, 2002). The application of enzymes such asheparinase, and N-acetylglucosamine-4-sulfatase, or arlysulfatases couldreduce the interaction and infection.

In a second scenario the pathogen molecular structure degraded could bea toxin that disrupts the target cell's metabolic functions. In a thirdscenario, the pathogenic component degraded by the enzyme might beinvolved in the pathogen's mechanism to evade the host immune response.Numerous immune response evasion mechanisms have evolved in pathogensranging from mimicking the host ells outer appearance to inhibitingimmune response, for example complement reactions or apoptosis. Thereduction or prevention of infection also can be assessed by measuringserum APP, with higher APP levels being associated with infection.

Enzymes that degrade pathogenic components, such as those describedabove, are known in the art. For example, an endosialidase derived froma bacteriophage was shown to prevent the lethality of E. coli K1systemic infection of rats by degrading the PSA (polysialic acid)capsule on the bacteria surface. Although degrading the capsularcarbohydrate has no effect on the viability of the E. coli in vitro,loss of capsule in vivo allows recognition and control of the infectionby the host immune system eliminating lethality (Mushtaq, N., et al.Antimicrobial Agents and ChemoTherapy 48(5):1503-1508, 2004). The PSAcapsule allows the E. coli surface to look like a host cell thus evadinghost innate immune responses. Another known enzyme useful in the presentinvention is heparinase I (Neutralase™, Ibex Technologies, Canada). Manyenzymes are available from commercial sources or can be obtained frommicroorganisms that produce enzymes, such as bacteria, and fungiincluding yeast, or can be produced recombinantly, as discussed above.

Desired enzymes can be produced by recombinant DNA techniques when thegene coding for the enzyme is known. The advancement of rapid DNAsequencing methodology has resulted in large public databases ofproteins and their gene coding sequences, such as the NCBI Genbank.Using rapid sequencing technology from, for example, 454 Life Sciences(454 Life Sciences, 20 Commercial Street, Branford, Conn. 06405), atypical bacterial genome can be sequenced in four hours. A previouslyunknown gene of new desired enzyme from the genome can be obtained byprobing the genome using, for example, previously identified codingsequences from the same type or similar types of enzymes described incommercial or public databases, using readily available computerprograms such as Blast. Those skilled in the art can identify DNA in thegenome that has a threshold level of homology to the known sequence andother properties of a gene-coding region, and then isolate and amplifythe gene using, for example, polymerase chain reaction (PCR) technology.The gene can then be expressed in a host and its desired proteinenzymatic properties can be confirmed.

If a desired enzyme activity is not previously known, then it can belocated using standard microbiology enrichment techniques selecting forgrowth on the substrate. Microbes using the substrate as the sole carbonor nitrogen source must express enzymes capable of degrading the targetcompound. In order to develop economical production, one has the choiceto improve the production of that enzyme using classicalmutation/selection or enrichment methods with the producingmicroorganism, or through recombinant DNA expression methods well knownin the art.

The composition comprising an immune stress-reducing enzyme thatdegrades a pathogenic microorganism may be any composition suitable foradministration to an animal, including compositions suitable for oraladministration to an animal, as described above. As noted above, thecomposition may comprise an amount of enzyme that is effective todecrease the level of positive acute phase protein (or increase thelevel of negative acute phase protein) in the animal and/or improveanimal growth performance. This amount may vary from animal to animal,and from enzyme to enzyme, but readily can be determined by thoseskilled in the art, for example, by measuring APP levels and/ormonitoring animal growth performance, as described above and as known inthe art.

In one embodiment, the immune stress-reducing enzyme that targets apathogenic antigen is provided as a component of an animal feed. In oneexample of this embodiment, the amount of enzyme is at least about100,000 IU/ton feed.

In another embodiment, the immune stress-reducing enzyme that targets apathogenic antigen is provided as a component of a composition that alsocomprises the pathogenic antigen. For example, the invention includes ananimal feed comprising (A) a pathogenic microorganism displaying anantigen such as lipopolysaccharide, peptidoglycan, lipotechoic acid,phosphorylcholine, double-stranded RNA and DNA and (B) and enzyme thatdegrades the antigen. Pathogenic organisms can find the way into feeddue to the inherent nature of unsanitary conditions caused by the densegrowth of animals in production situations.

As noted above, the invention includes methods of reducing immune stressin an animal and/or of improving aimal growth performance, comprisingadministering to the animal a composition comprising an immunestress-reducing enzyme. In one embodiment, the animal is administeredthe immune stress-reducing enzyme that degrades a pathogenic antigen andalso is administered the pathogenic antigen. The enzyme and antigen maybe administered separately or simultaneously as part of the same ordifferent compositions. In one embodiment, the animal is administered afeed comprising the antigen, and is separately administered acomposition comprising the enzyme. In another embodiment, the animal isadministered a feed comprising the antigen and a feed supplementcomprising the enzyme. In another embodiment, the animal is administereda feed comprising both the antigen and the enzyme.

The following examples further illustrate the invention, but theinvention is not limited to the specifically exemplified embodiments.

Example 1

An animal feed comprising hemicellulase (endo-1,4-β-mannanase) wasprepared and administered to chickens and AGP levels were measured, asdescribed in more detail below.

A total of 4000 one-day-old male Cobb X Cobb chicks were allocated atrandom to 8 experimental treatments, and each treatment was replicated10 times:

Experimental Design: 8 Treatments Total No. of pens: 80 Total No. ofTreatments: 8 No. of birds per pen: 50 No. of pens per Treatment: 10 No.of birds per Treatment: 500

Two of the eight treatments comprised stress-reducing enzymes inaccordance with the invention: Treatment 3 (mannanase in the form ofevaporated whole cell broth of B. lentus fermentation applied atapproximately 100 MU/ton in the basal diets) and Treatment 6 (mannanasein the form of cell-free centrifuged supernatant of B. lentusfermentation broth applied at approximately 30 MU/ton in the basaldiets). Treatment 8 was a control with no added enzyme. (1 MU=4000 IU)

The basal meal feed batches were divided evenly in eight parts and eachwas sprayed with the appropriate amount of the test materials. Starterand Grower feeds contained 90 g/ton Cobon™ (an anticoccidial drug of theionophore type) plus 50 g/ton BMD® (antibiotic). Finisher feeds werenon-medicated.

Starter Diets were offered to all birds from day-old until 21 days ofage, Grower Diets from 22-35 days, and Finisher Diets from 36-42 days.The diets and water were provided ad libitum. The diets were presentedto the birds as crumbles/pellets during all feeding periods. Tap waterwas used as drinking water and supplied by an internal water systemnetwork.

Composition and Analyses of the Basal Experimental Diets

Ingredients Starter Grower Finisher Corn 60.3851 67.6864 72.1098 Soybeanmeal (48.5% CP) 34.5066 27.8363 23.3785 Fat AV Blend 1.0516 0.99151.1389 Dicalcium phosphate 1.761 1.2682 1.3021 Limestone flour 1.31921.383 1.26 Sodium chloride 0.3299 0.3304 0.3305 DL Methionine 0.21350.0793 0.0552 L-lys.hcl 0.008 — — Choline chloride 70% 0.05 0.05 0.05Vitamin premix 0.25 0.25 0.25 Mineral premix 0.075 0.075 0.075 Coban,g/ton 90 90 — BMD, g/ton 50 50 — Calculated Analyses² ME_(n) poultry(kcals/kg) 3080.0 3150.0 3200.0 Dry matter, % 88.9169 88.9236 88.9054Crude protein, % 22.0 19.3 17.5 Crude fibre, % 2.8813 2.8176 2.7632 Fat,%, 3.6777 3.8291 4.0981 Calcium, % 1.0 0.9 0.85 Total phosphorus, %0.7088 0.5967 0.5877 Available phosphorus, % 0.45 0.35 0.35 Sodium, %0.18 0.18 0.18 Lysine, % 1.2 1.0152 0.8948 Methionine + Cysteine, % 0.920.72 0.65 Threonine, % 0.8821 0.7657 0.6932 Tryptophan, % 0.2938 0.24890.2185

Two birds from each of the ten pens in Treatments 3, 6 and 8 wererandomly selected for blood analysis at the end of the 42 days afterweighing, for a total of 20 birds out of the 500 per treatment. Sampleswere collected onto ice in blood collection tubes containinganti-coagulant heparin, and plasma was obtained by centrifugation.

Blood plasma samples were assayed for chicken α-1-acid glycoproteinusing an immunodiffusion based assay kit from Cardiotech Services, Inc.(Louisville, Ky.). Serum samples taken from the two birds/pen were addedto the test plates (5 μL per well) and to some wells standard pure AGPwas added at concentrations ranging up to 1000 μg/mL. Precipitin ringswere measured using a precipitin ring measurement scale to the nearest0.1 mm diameter.

A polynomial equation was used to provide the best curve fit with thedata and to allow the rapid calculation of the concentration of AGP inthe plasma samples as shown in FIG. 1.

The measurements of precipitin ring diameters for all the recoveredchicken serum samples and the calculated AGP concentration for each birdis shown in the table below. The birds fed mannanase on average have avery statistically significant decrease in the average AGP concentrationcompared to the control birds.

42 Day Blood Samples (y=AGP ug/ML; x=ring measurement in mm)

Treatment 3 Treatment 8 Treatment 6 (Mannanase) (control) (Mannanase) Xy x y X y 5.3 225.0 6 317.7 5.3 225.0 5.3 225.0 6 317.7 5.7 276.2 4.9178.6 6.1 332.1 5.8 289.7 5.2 212.9 5.2 212.9 5.1 201.2 5.8 289.7 7.4546.9 5.4 237.4 5.4 237.4 5.4 237.4 5.7 276.2 5.4 237.4 6.2 346.9 5.5250.0 5.9 303.6 5.9 303.6 6.1 332.1 5.6 262.9 6 317.7 5.4 237.4 5.9303.6 6.2 346.9 5.2 212.9 5.2 212.9 6.1 332.1 5 189.7 5.4 237.4 5.6262.9 6.1 332.1 5.3 225.0 5.9 303.6 5.5 250.0 5.1 201.2 6.3 361.9 5.5250.0 5.3 225.0 6.2 346.9 6 317.7 5.3 225.0 7.5 565.5 5.4 237.4 5.7276.2 7.4 546.9 5.6 262.9 5.3 225.0 7.5 565.5 4.4 127.2 5.7 276.2 7.5565.5 5   189.7 6.1 332.1 AVE 238.5 373.1 250.3 SD 35.8 115.6 51.3 CV14.99 30.97 20.50 T Test p vs. 8 2.94E−05 T Test vs. 8 0.000193

Example 2

Another experiment using hemicellulase (endo-1,4-β-mannanase) wasconducted. In this experiment, groups of chickens (10 pens each, with 50birds per pen) were fed one of four diets:

Treatment 1 (control): Feed comprising BMD antibiotic sprayedpost-pelleting with a control formulation, and 35% sorbitol with brownfood dye, applied at 100 ml/ton feed.

Treatment 2 (control): Feed without BMD sprayed post-pelleting with acontrol formulation comprising 35% sorbitol with brown food dye, appliedat 100 ml/ton feed.

Treatment 3: Feed sprayed post-pelleting with a formulation comprisinghemicellulase (endo-1,4-β-mannanase) derived from B. lentus, applied at100 ml/ton feed.

Treatment 4: Feed formulated with a powder composition (added into themixer prior to pelleting) comprising hemicellulase(endo-1,4-β-mannanase) derived from B. lentus at 454 g of compositionadded/ton feed to provide 100 MU/tom of feed. (1 MU=4000 IU)

The chickens were 1 day old at the start of the experiment.

The diets were provided ad libitum. Starter (days 0-21), grower (days21-35) and finisher (days 35-42) feeds with the following compositionswere used as the base feeds:

Expected Amounts

Nutrient Analysis Starter Grower Finisher ME POUL KCAL 2960.0 3020.03080.0 Crude Protein 22.0 19.4 17.5 Fat % 3.1439 3.0647 3.4503 Calcium %0.9 0.8 0.8 T phos 0.7032 0.6315 0.515 A Phos 0.45 0.39 0.35 Sodium 0.180.18 0.18 Lysine % 1.205 1.0302 0.9014 Methionine 0.5446 0.3838 0.3435Met + Cys 0.92 0.72 0.65

Added Ingredients

Ingredient Starter Grower Finisher Limestone 0.8291 0.7674 0.9012 Salt0.2696 0.2698 0.2702 D-L Meth 0.1963 0.0682 0.0532 Choline Chloride 70%0.0500 0.0500 0.0500 Dical P 1.6869 1.4088 1.2295 Fat 0.6517 0.47510.8162 Corn 59.3480 67.4725 70.9038 Soybean meal 33.5934 27.1132 22.4010Poultry By-Product 3.0 3.0 3.0 meal Vitamin 0.25 0.25 0.25 Mineral 0.0750.075 0.075 Salinomycin 0.05 0.05 0.05

On day 21 approximately 3 ml of blood were collected from 3 birds perpen (30 per group). Blood was placed into a heparinized tube and lightlymixed. Tubes were slowly centrifuged and then serum was removed. Serumsamples were placed into tubes with caps and labeled with pen number.Serum was frozen for subsequent AGP analysis, as described in Example 1above. The immunodiffusion rings used to quantitate chicken al acidglycoprotein are easily measured, highly reproducible and exhibit acoefficient of variation of 4% or less.

The 21 day average results of 30 birds per treatment are shown in thetable below and graphically in FIG. 2. It can be seen that leaving theantibiotic (BMD) out of the diet creates a large and significantincrease in the plasma AGP level (compare Treatment 1 and Treatment 2).The addition of either hemicellulase (endo-1,4-β-mannanase) formulationinto the no-BMD diet (Treatments 3 and 4) restored the AGP to the levelseen with antibiotic use, indicating a significant reduction ofimmunological stress.

Treatment 1 2 3 4 AGP Avg. 214.35 267.99 220.28 233.09 SD 62.16 82.4268.58 67.73 CV 29.00 30.76 31.13 29.06 TTest P 0.003055 0.008952 0.03919vs. Trt. 1 Trt. 2 Trt. 2 TTest P 0.234892 vs. Trt. 3 TTest P 0.3633050.134383 vs. Trt. 1 Trt. 1

The growth performance of the chickens also was assessed, with theresults summarized in the table below.

Growth Performance

Day 21 FCR¹ P val. Wt. gain P val. Wt. adj. FCR² P val. CV of ID³ P val.T1 1.394 0.150 0.693 0.016 1.379 0.017 13.81 0.33 T2 1.412 0.657 1.42414.77 T3 1.404 0.605 0.673 0.197 1.404 0.248 14.03 0.46 T4 1.407 0.7400.670 0.459 1.410 0.551 14.40 0.69 Day 42 T1 1.776 0.006 2.102 0.1891.772 0.007 11.23 0.19 T2 1.813 2.073 1.820 10.42 T3 1.770 0.001 2.1310.088 1.756 0.005 9.97 0.49 T4 1.761 0.0001 2.060 0.572 1.772 0.00310.38 0.95 ¹FCR = Feed conversion ²Wt. Adj. FCR = weight adjusted feedconversion ³CV of ID = coefficient of variation in individual weights

Thus, both feed conversion and weight adjusted feed conversion wereimproved at 21 days with statistical significance in chickens receivingβ-mannanase. This indicates that the reduction in serum AGP cantranslate into real significance for animal performance.

Example 3

The ability of other enzymes commonly used in animal feed were assessedfor their possible effect on AGP. Commercial type chicken starterrations (low metabolic energy) were compounded with feedstuffs commonlyused in the United States. These rations (in mash or crumble form) werefed ad libitum from the date of chick arrival until Day 21 of the study.Experimental treatment feeds were prepared from this basal starter feed.Treatment feeds were mixed to assure a uniform distribution ofrespective test article.

Composition and Analyses of the Basal Experimental Diets

Ingredients Corn 59.398 Soybean meal (48.5% CP) 33.5934 Fat AV Blend0.6517 Dicalcium phosphate 1.6869 Limestone flour 0.8291 Sodium chloride0.2696 DL Methionine 0.1963 Poultry By Product Meal 3.0 Choline chloride70% 0.05 Vitamin premix 0.25 Mineral premix 0.075 LO ME ME_(n) poultry(kcals/kg) 2960 Crude protein, % 22.0 Crude fibre, % 2.8899 Fat, %,3.1439 Calcium, % 0.9 Total phosphorus, % 0.7032 Available phosphorus, %0.45 Sodium, % 0.18 Lysine, % 1.205 Methionine + Cysteine, % 0.92Threonine, % 0.8266BMD 50 g/t and Salinomycin 60 g/t were added to all feeds.

Enzyme Preparations

Sample enzyme assay data Stock vol Diluent Notes Use Level 3 — n.d. 20plus food  20 mL/100 Kg coloring 6 Adessio, Rovabio 10 10  20 mL/100 KgRovabio commercial Excel LC product 7 Danisco, Avizyme Solid — 100 g/100Kg Avizyme commercial product (1500 product Granular) 8 PI-PLC  106 U/mL50 — 1.0 mL/Kg 9 Genencor  20 mL/100 Kg Amylase FRED 10 Genencor  20mL/100 Kg Multifect XL 11 Dyadic  20 mL/100 Kg Brewzyme BG 12 Hemicell1092 MU/L 13 12  20 mL/100 Kg 17 — n.d. 20 plus food  20 mL/100 Kgcoloring (1 MU = 4000 IU)

Enzyme Preparations Further Information

Endo-1,3 β-glucanase Sample Main Activity Use Level minor activity* 3 —6 Endo-1,4-β- Endo-1,4-β-Xylanase 9139 IU/ton Xylanase 350 AXC 350 AXCU/Ml 63,350 AXC U/ton Endo β-1,4- Endo β-1,4- β-glucanase β-glucanase500 AGL U/mL 90,500 AGL U/ton 7 Amylase 1.0 kg per ton — xylanaseprotease 8 PI-PLC 96,188 IU/ton or — 106,000 IU/L 24 MU/ton 9 Amylase4700 MU/L 1.88 × 10⁶ IU/ton or — 470 MU/ton 10 Endo-1,4-β- 900,000IU/ton or — Xylanase 225 MU/ton 4500 MU/L 11 Endo β-1,4-1,3- 634,400IU/ton or 1040 IU/ton glucanase 159 MU/ton 1586 MU/L 12 Endo-1,4 β-400,000 IU/ton or  580 IU/ton mannanase 100 MU/ton 1092 MU/L 17 —ChemGen MU = 4000 IU; AXC - xylanase units defined by Adisseo; AGL -glucanase units defined by Adisseo; *approximate level measured byChemGen Corp. by reducing sugar assay

Feed and water were available ad libitum throughout the trial. On Day15, birds in treatment 17 were orally inoculated with a mixed inoculumcontaining approximately 30,000 oocysts E. acervulina per bird, 2,500oocysts of E. maxima per bird, and 25,000 oocysts E. tenella per bird.Coccidial oocyst inoculation procedures are described in SPR SOP:IN1.002.

Means for cage weight gain, feed consumption, and feed conversion aredetermined. The results are set forth below. Only animals receivingsample 17 were infected.

Vs. Treatment 3 21 Day Growth Data TTEST Avg. Enzyme Sample P= TreatmentAGP ave Live Wt. Gain Conv. level per metric Ton 3 — control 170.520.624 1.438 none 6 0.2076 Rovabio 186.55 0.626 1.395 100 mL 7 0.2770Avizyme 160.44 0.633 1.426 1.0 kg 8 0.1263 PI-PLC 196.35 0.650 1.406106,000 IU 9 0.3962 Amylase 164.05 0.622 1.434 10 0.3783 Xylanase 175.890.593 1.444 11 0.2647 Glucanase 182.00 0.629 1.396 12 0.0178 Hemicell138.22 0.645 1.421 102 MU 17 0.0043 control- 252.04 0.564 1.507 noneinfected

We found that commercial feeds comprising standard amounts of amylase,1,3-glucanase, 1,4-glucanase, xylanase and PI-PLC did not reduce AGPlevels. Indeed, only hemicellulase (endo-1,4-β-mannanase) showed asignificant effect on AGP levels. Additionally, comparing treatment 1and 17 clearly shows that AGP is a highly responsive APP in chickensbecause infection increased the AGP level 82 μg/mL. See also FIG. 3.

Example 4

Test animal feed comprising 1,3-β-glucan is formulated to include1,3-β-glucanase at a concentration of 400,000 IU (100 ChemGen MU) perton feed. The test animal feed is administered to test chickens, whilecontrol chickens receive the same animal feed (comprising 1,3-β-glucan)without 1,3-β-glucanase. After 21 and 42 days on this regimen, bloodserum AGP levels are assessed as described above. Chickens receiving theenzyme-formulated animal feed have significantly lower levels of AGPthan control animals. The test chickens also exhibit greater feedefficiency and improved weight gain as compared to control chickens.

Example 5

Test animal feed comprising a source of bacterial DNA (e.g. Biolys®Lysine or other fermentation product containing cell products) isformulated to include non-specific nuclease derived from theCyanobacterium Anabaena sp. 7120 (NucA), one of the most activenon-specific nucleases known (Meiss, G. et al., Eur. J. Biochem. 251(3):924-934, 1998). The enzyme is added at a concentration of 1×10⁷ KunitzUnits enzyme/kg feed or approximately 1 mg (pure basis) per kg of feed.The test animal feed is administered to test chickens, while controlchickens receive the same animal feed (comprising bacterial DNA) withoutnon-specific nuclease. After 21 days or 42 days on this regimen, bloodserum AGP levels are assessed as described above. Chickens receiving theenzyme-formulated animal feed have significantly lower levels of AGPthan control animals.

Example 6

Test animal feed comprising meat and bone meal, blood meal or otheranimal derived by-product is formulated to include phosphatidylserinedecarboxylase at a concentration of 400,000 IU/ton of feed. The testanimal feed is administered to test chickens, while control chickensreceive the same animal feed without phosphatidylserine decarboxylase.After 21 days or 42 days on this regimen, blood serum AGP levels areassessed as described above. Chickens receiving the enzyme-formulatedanimal feed have significantly lower levels of AGP than control animals.

Example 7

Test animal feed soy meal or other plant derived meal is formulated toinclude an α-mannanase and/or 1,3-β-glucanase enzymes derived from B.lentus, each at a concentration of 400,000 IU/ton of feed. The testanimal feed is administered to test chickens, while control chickensreceive the same animal feed without α-mannanase or 1,3-β-glucanase.After 21 days or 42 days on this regimen, blood serum AGP levels areassessed as described above. Chickens receiving the enzyme-formulatedanimal feed have significantly lower levels of AGP than control animals.

Example 8

In this example Hemicell® mannanase added to feed (a conventionalcorn-soybean diet) was shown to reduce al acid glycoprotein (AGP) inturkey serum while also improving live growth performance. Theexperiment consisted of 48 pens of 11 tom turkeys (initial placement).The six treatments were replicated in 8 blocks, randomized within blocksof six pens each:

No. Birds/Treatment 88 No. Reps/Treatment 8 Total Treatments 6 Total No.Pens 48 Total No. Birds 528

One treatment that comprised stress-reducing enzymes in accordance withthe invention, Treatment 1 (commercial Hemicell® with 100 MU/ton offeed) was analyzed for AGP. (1 MU=4000 IU) Treatment 2 was a controlfeed without added enzyme.

Feed was mixed to assure uniform distribution of basal feeds amongtreatments. All enzymes were mixed (sprayed on) to assure a uniformdistribution of test enzymes and to assure similar feed conditionbetween treatments. Each time treatment feed was made, a sample from thebeginning, middle, and end of each treatment feed were mixed to form acomposite sample. One sample was taken from the composite for eachtreatment, and for enzyme level verification.

The turkey diets fed in this study to Treatments 1 and 2 are describedin detail below in the following tables. Tables show the composition ofcomponents, the calculated nutrient levels and finally some measurednutrient values with the returned feeds. The diets are representative ofwhat might be used in a commercial turkey growing operation and thus thediet is adjusted several times throughout the 20-week period. Dietcompositions were changed at 6, 9, 12, 15 and 18 weeks.

The diet compositions at each period are slightly different forTreatments 1 and 2. It is well known that Hemicell® mannanase has theeffect of increasing the effective energy content of feeds (see U.S.Pat. No. 6,162,473). For that reason, the diets of Treatment 1 areformulated with fewer calories than the diets of Treatment 2 in order tominimize growth difference between Treatments 1 and 2 for the purpose ofthis study.

Ingredient Composition and Calculated Nutrient Levels, 0-9 Weeks

Period: 0-6 weeks 6-9 weeks Treatment 1 Treatment 1 with Treatment withTreatment Ingredient (%) Hemicell ® 2 Hemicell ® 2 Corn 46.77 45.3953.14 51.79 Soybean meal 37.15 37.40 29.30 29.50 Poultry meal 9.00 9.009.00 9.00 Poultry Fat 1.50 2.65 3.50 4.65 Limestone 1.20 1.20 1.25 1.25Dical phosphate 2.70 2.70 2.35 2.35 18.5 Salt 0.325 0.325 0.315 0.32 DLMethionine 0.315 0.315 0.245 0.25 L-Lysine-HCl 0.41 0.405 0.335 0.34Vitamin pre-mix 0.25 0.25 0.25 0.25 Trace minerals 0.075 0.075 0.0750.075 Choline Cl 60% 0.135 0.135 0.085 0.085 Copper sulfate 0.05 0.050.05 0.05 Coban 60 g/lb 0.055 0.055 0.05 0.05 BMD 50 g/lb 0.05 0.05 0.050.05 Hemicell ® 0.0125 0.0 0.0125 0.0 Crude protein (%) 28.00 28.00 24.524.5 ME (Kcal/lb) 1323 1323 1408 1407 Calcium (%) 1.484 1.484 1.4621.462 A. Phosphorus (%) 0.797 0.797 0.764 0.763 Lysine (%) 1.794 1.7931.502 1.501 Met + Cys (%) 1.179 1.177 1.018 1.021

Ingredient Composition and Calculated Nutrient Levels, 9-15 Weeks

Period: 9-12 weeks 12-15 weeks Treatment 1 Treatment 1 with Treatmentwith Treatment Ingredient (%) Hemicell ® 2 Hemicell ® 2 Corn 56.88 55.5562.45 61.05 Soybean meal 24.55 24.75 21.15 21.40 Poultry meal 9.00 9.007.00 7.00 Poultry Fat 5.00 6.22 5.00 6.15 Limestone 1.20 1.20 1.15 1.15Dical phosphate 1.95 1.95 1.75 1.75 18.5 Salt 0.32 0.32 0.32 0.32 DLMethionine 0.22 0.22 0.30 0.30 L-Lysine-HCl 0.315 0.315 0.42 0.42Vitamin pre-mix 0.25 0.25 0.25 0.25 Trace minerals 0.075 0.075 0.0750.075 Choline Cl 60% 0.085 0.085 0.015 0.015 Copper sulfate 0.05 0.050.05 0.05 Coban 60 g/lb 0.05 0.05 0.00 0.00 BMD 50 g/lb 0.05 0.05 0.050.05 Hemicell 0.0125 0.0 0.0125 0.0 Crude protein (%) 22.5 22.5 20.020.0 ME (Kcal/lb) 1469 1469 1490 1490 Calcium (%) 1.35 1.35 1.19 1.19 A.Phosphorus (%) 0.681 0.681 0.59 0.59 Lysine (%) 1.350 1.350 1.298 1.298Met + Cys (%) 0.940 0.940 0.95 0.95

Ingredient Composition and Calculated Nutrient Levels, 15-20 Weeks

Period: 15-18 weeks 18-20 weeks Treatment 1 Treatment 1 with Treatmentwith Ingredient (%) Hemicell ® 2 Hemicell ® Treatment 2 Corn 67.25 65.8570.60 69.15 Soybean meal 17.90 18.15 15.60 15.85 Poultry meal 5.00 5.004.00 4.00 Poultry Fat 6.00 7.15 6.50 7.70 Limestone 1.00 1.00 0.85 0.85Dical phosphate 1.50 1.50 1.23 1.23 18.5 Salt 0.33 0.33 0.34 0.34 DLMethionine 0.205 0.205 0.193 0.193 L-Lysine-HCl 0.340 0.340 0.235 0.235Vitamin pmx 0.25 0.25 0.25 0.25 Trace minerals 0.075 0.075 0.075 0.075Choline Cl 60% 0.02 0.02 0.02 0.02 Copper sulfate 0.05 0.05 0.05 0.05Coban 60 g/lb 0.00 0.00 0.00 0.00 BMD 50 g/lb 0.05 0.05 0.05 0.05Hemicell 0.0125 0.0 0.0125 0.0 Crude protein (%) 17.5 17.5 16.0 16.0 ME(Kcal/lb) 1539 1539 1570 1570 Calcium (%) 0.982 0.982 0.82 0.82 A.Phosphorus 0.490 0.490 0.41 0.41 (%) Lysine (%) 1.10 1.10 0.93 0.93Met + Cys (%) 0.791 0.791 0.74 0.74Diet Analysis with Returned Feeds, 0-12 Weeks

Treatment 1 with Hemicell ® Treatment 2 Nutrient Calculated AnalyzedCalculated Analyzed 0-3 weeks Protein 28 27.46 28 27.57 Fat 4.2 4.03 5.34.99 Calcium 1.48 1.38 1.48 1.45 T. Phosphorus 1.02 0.96 1.02 1.08Hemicell units 100 70.1 0 8.9 3-6 weeks Protein 28 25.90 28 27.17 Fat4.2 4.01 5.3 5.16 Calcium 1.48 1.63 1.48 1.52 T. Phosphorus 1.02 1.151.02 1.12 Hemicell units 100 99.3 0 8.9 6-9 weeks Protein 24.5 23.4224.5 24.17 Fat 6.3 6.33 7.4 7.00 Calcium 1.46 1.69 1.46 1.65 T.Phosphorus 0.96 1.09 0.96 1.06 Hemicell units 100 138.6 0 14.4 9-12weeks Protein 22.5 22.61 22.5 23.61 Fat 7.9/8.0 7.91 9.0 9.02 Calcium1.35 1.37 1.35 1.32 T. Phosphorus 0.86 0.93 0.86 0.90 Hemicell units 10083.9 0 12.0Diet Analysis with Returned Feeds, 12-20 Weeks

Treatment 1 with Hemicell ® Treatment 2 Nutrient Calculated AnalyzedCalculatd Analyzed 12-15 weeks Protein 20 20.49 20 21.09 Fat 7.79 8.258.89 9.03 Calcium 1.19 1.16 1.19 1.09 T. Phosphorus 0.76 0.78 0.76 0.77Hemicell units 100 90.5 0 13.6 15-18 weeks Protein 17.5 17.20 17.5 16.24Fat 8.68 8.00 9.78 9.21 Calcium 0.98 0.96 0.98 0.94 T. Phosphorus 0.650.68 0.65 0.70 Hemicell units 100 98.6 0 5.6 18-20 weeks Protein 16.015.95 16.0 15.14 Fat 9.14 8.93 10.29 10.39 Calcium 0.82 0.79 0.82 0.87T. Phosphorus 0.57 0.61 0.57 0.65 Hemicell units 100 113.4 0 13.6

Glycoprotein Measurement:

Blood was obtained at the end of the trial from four birds per penselected at random from treatments 1,2, and 5. The blood was collectedinto tubes containing EDTA anticoagulant, mixed then centrifuged toprecipitate the whole cells.

Turkey AGP test plates were obtained from Cardiotech Services(Louisville, Ky.). The AGP test is an immunodiffusion based test. Equalvolumes of test or serum samples were added into the immunodiffusionplate wells as recommended by the manufacturer, then after two daysincubation at room temperature, the diameter of the resultingimmunoprecipitation rings was measured.

A sample of purified turkey AGP standard provided in the kit was testedat several concentrations to make a standard curve as shown in FIG. 4. Apolynomial curve fit equation developed from the standards was used tocalculate the turkey plasma AGP level in the test samples.

Calculation of AGP Levels and Statistical Analysis with Students T Test

Hemicell mannanase (Treatment 1) Control (Treatment 2) mm AGP outlier mmAGP outlier 5.2 231.5 5.7 304.5 4.8 181.6 5.1 218.4 5 205.7 5.6 288.9 5205.7 5.3 245.1 5 205.7 5.1 218.4 5.1 218.4 5.1 218.4 5.3 245.1 5.9337.3 6.1 372.3 5 205.7 5.8 320.6 6.3 409.6 4.7 170.2 7.5 687.1 4.9193.4 6.45 439.1 5 205.7 5.4 259.2 5.2 231.5 5.1 218.4 5.5 273.8 5.3245.1 5.7 304.5 5.1 218.4 5.1 218.4 5.6 288.9 4.7 170.2 5.6 288.9 5205.7 6.05 363.3 4.5 148.5 5.6 288.9 5.3 245.1 5.4 259.2 5.2 231.5 5205.7 4.4 138.3 5 205.7 4.3 128.4 5.2 231.5 6.35 419.3 5.4 259.2 4.9193.4 4.8 181.6 5.65 296.6 5 205.7 5.2 231.5 5.2 231.5 5 205.7 5.4 259.24.8 181.6 4.9 193.4 5.2 231.5 5.1 218.4 7.5 687.1 4.2 118.9 5 205.7 6.5449.3 Ave 226.4 260.5 Std. 63.4 74.6 Deviation CV 28.0 28.6 T Test Pvalue 0.0284 Outliers >2 std. deviation from mean removed

The average plasma AGP for the enzyme treated group was significantlyless than the untreated control. For this analysis, one outlier wasremoved from the analysis from each group. These may be birds thatexperienced an unusual amount of stress due to injury or infection. Thereduced AGP caused by enzyme feeding was correlated with statisticallysignificant improved live bird performance as shown below in Treatment 1(mannanase) vs. Treatment 2.

140 Day Growth Results Hemicell Feed Live CV in weight Pen Weight Mort.# Mort Weight Consumed Feed Conv. Wt. Gain 140 d 3 200.25 0 0 487.352.434 18.147 5.766 7 193.3 0 0 479.55 2.481 17.512 7.426 14 143.95 310.817 389.05 2.514 17.934 6.857 21 164.1 2 24.205 448.70 2.383 18.1724.411 29 185.8 1 0.975 448.90 2.403 18.521 6.092 36 161.1 2 16.49 419.902.364 17.839 8.352 37 182.1 1 7.611 443.70 2.339 18.150 5.944 47 182.551 13.115 449.05 2.295 18.195 3.405 Avg. 445.78 2.402 18.059 6.032 T TestP val 0.05 0.01 0.05 Vs. Trt 2 Control 5 144.1 2 13.085 389.00 2.47515.950 14.518 12 196.1 0 0 486.70 2.482 17.766 5.288 13 178.7 1 14.65466.25 2.411 17.810 9.717 23 141.95 3 21.912 389.40 2.376 17.684 8.01228 190.2 0 0 482.10 2.535 17.231 9.331 35 194.5 0 0 486.25 2.500 17.6229.722 42 159.25 2 10.814 409.40 2.407 17.635 5.054 48 176.65 1 9.05459.10 2.472 17.605 4.772 Avg. 446.03 2.457 17.413 8.302

Birds receiving feed with mannanase had greater average weight gain by3.7%, decreased feed conversion by 2.3% and decrease in the CV(coefficient of variation=std. deviation/mean) of body weightuniformity. The reduction in immune stress as indicated by reduced AGPserum levels correlated with several measurements of growth improvement.

Example 9

In this example 1,4-β-mannanase from B. lentus, 1,3-β-glucanase from B.lentus, and a combination of the two enzymes were added to feed (aconventional corn-soybean diet). Each enzyme treatment improved livegrowth performance in 6 week old Nicholas 700 female turkeys, withresults achieved by the combination being unexpectedly greater thanresults achieved with treatments using only one of the enzymes.

The experiment used 80 pens of 40 female turkeys. The treatments werereplicated in ten (10) blocks, with eight treatments (seven enzymetreatments and one negative control) randomized within each block.

Treatment 3 used a composition comprising an immune stress-reducingenzyme in accordance with the invention, 1,4-β-mannanase at 100 MU/tonof feed. Treatment 6 also used a composition comprising an immunestress-reducing enzyme in accordance with the invention, 1,3-β-glucanaseat 60 MU/ton of feed. Treatment 8 used a combination composition inaccordance with the invention, comprising 1,4-β-mannanase at 100 MU/tonof feed and 1,3-β-glucanase at 60 MU/ton of feed. Treatment 1 was acontrol feed without added enzyme. (1 MU=4000 IU)

Feed was mixed to assure uniform distribution of basal feeds amongtreatments. All enzymes were mixed (sprayed on) to assure a uniformdistribution of test enzymes and to assure similar feed conditionbetween treatments. Each time a treatment feed was made, a sample fromthe beginning, middle, and end of each treatment feed were mixed to forma composite sample. One sample was taken from the composite for eachtreatment, and for enzyme level verification.

The turkey diets fed in this study are typical commercial turkey feeds.The diets are representative of what might be used in a commercialturkey growing operation and thus the diet was adjusted after 3 weeks.The growth results for Treatments 1, 3, 6 and 8 are shown in the tablebelow.

6-week Growth Parameters Average Weight- Mor- Live Adjusted talityWeight Feed Feed Treatment (%) (lbs) Conversion¹ Conversion² T1 Control1.75^(A) 5.406^(A) 1.500^(A) 1.520^(A) T3 1,4-β-mannanase 1.50^(A)5.484^(ABC) 1.495^(AB) 1.502^(AB) (100 MU/ton) T6 1,3-β-glucanase1.50^(A) 5.540^(C) 1.467^(C) 1.465^(C) (60 MU/ton) T8 1,4-β-mannanase3.25^(A) 5.718^(D) 1.420^(D) 1.389^(D) (100 MU/ton) and 1,3-β-glucanase(60 MU/ton) (1 MU = 4000 IU) Note ¹Feed Conversion is mortalitycorrected. Note ²Weight-adjusted feed conversion for each treatment iscalculated as follows: (a) the average live weight of the entire test issubtracted from the average live weight for the treatment, resulting inQuantity A; (b) Quantity A is divided by 6, resulting in Quantity B; (c)Quantity B is subtracted from the feed conversion resulting in theweight-adjusted feed conversion for the treatment. Statistics shown arefor LSD test; P < 0.05.

In comparison to Treatment 1 (control), Turkey hens receiving Treatment3 (feed with 1,4-β-mannanase) had a numerically improved average liveweight and a numerically improved (decreased) feed conversion.Similarly, Turkey hens receiving Treatment 6 (feed with 1,3-β-glucanase)had a statistically-significant improved average live weight and astatistically-significant improved (decreased) feed conversion.

Surprisingly, Turkey hens receiving Treatment 8 (feed with a combinationof 1,4-β-mannanase and 1,3-β-glucanase) had an unusually largestatistically significant improved average live weight and an unusuallylarge statistically significant improved (decreased) feed conversion.The results observed with Treatment 8 were greater than could beexplained by an additive effect of the two enzymes administeredindividually. Thus, the combination treatment comprising 1,4-β-mannanaseand 1,3-β-glucanase produced an unexpectedly large improvement in growthperformance.

Example 10

In this example, 1,3-β-glucanase from B. lentus, xyloglucanase from B.lentus and a combination of the two enzymes were added to feed (aconventional corn-soybean diet). Each enzyme treatment improved livegrowth performance in 35-day old male broiler chickens, with resultsachieved by the combination being greater than results achieved withtreatments using only one of the enzymes.

The experiment used 49 pens of 44 Cobb x Cobb male chickens. Thetreatments were replicated in seven blocks, with seven treatments (sixenzyme treatments and one negative control) randomized within eachblock.

Treatment 4 used a composition comprising an immune stress-reducingenzyme in accordance with the invention, 1,3-β-glucanase at 70 MU/ton offeed. Treatment 5 also used a composition comprising an immunestress-reducing enzyme in accordance with the invention, xyloglucanaseat 100 MU/ton of feed. Treatment 6 used a combination composition inaccordance with the invention, comprising xyloglucanase at 100 MU/ton offeed and 1,3-β-glucanase at 60 MU/ton of feed. Treatment 1 was a controlfeed without added enzyme. (1 MU=4000 IU).

Feed was mixed to assure uniform distribution of basal feeds amongtreatments. All enzymes were mixed (sprayed on) to assure a uniformdistribution of test enzymes and to assure similar feed conditionbetween treatments. Each time a treatment feed was made, a sample fromthe beginning, middle, and end of each treatment feed were mixed to forma composite sample. One sample was taken from the composite for eachtreatment, and for enzyme level verification.

The diets fed in this study are typical broiler chicken feeds. The dietsare representative of what might be used in a commercial broiler growingoperation and thus the diet was adjusted after 3 weeks. The growthresults for Treatments 1, 4, 5, and 6 after 35 days of growth are shownin the table below:

35-day Growth Parameters Weight- Mor- Average Adjusted tality LiveWeight Feed Feed Treatment (%) (lbs) Conversion¹ Conversion² T1 Control3.25^(A) 4.347^(A) 1.675^(A) 1.690^(A) T4 1,3-β-glucanase 3.25^(A)4.433^(AB) 1.651^(AB) 1.651^(AB) (70 MU/ton) T5 Xyloglucanase 2.92^(A)4.415^(AB) 1.646^(AB) 1.650^(AB) (100 MU/ton) T6 Xyloglucanase 4.55^(A)4.461^(AB) 1.643^(AB) 1.639^(AB) (100 MU/ton) And 1,3-β-glucanase (70MU/ton) (1 MU = 4000 IU) Note ¹Feed Conversion is mortality corrected.Note ²Weight-adjusted feed conversion for each treatment is calculatedas described above. Statistics shown are for LSD test; P < 0.05.

In comparison to Treatment 1 (control), the chickens receiving Treatment4 (1,3-β-glucanase) had a numerically improved average live weight and anumerically improved (decreased) feed conversion. Similarly, thechickens receiving Treatment 5 (feed with xyloglucanase) had anumerically improved average live weight and a numerically improved(decreased) feed conversion.

Chickens receiving Treatment 6 (a combination of 1,3-β-glucanase andxyloglucanase) achieved improvements in their average live weight andfeed conversions greater than the effect observed when the enzymes wereadministered individually. Thus, the combination treatment comprising1,3-β-glucanase and xyloglucanase achieved a significant improvement ingrowth performance.

Example 11

Reduction of Chicken Serum APP by Application of Enzymes in Feed

Chicken broilers were grown from 1 to 14 days and fed a typicalcorn-soybean starter diet (as shown in the Diet Composition table below)with various enzymes added (as summarized in the Enzyme table below).Sample sizes for each enzyme type included three cages with eight birdsper cage.

Diet Composition

Component Percent Corn 7.35% CP 53.98 Soybean meal 47.2 CP 39.03 Soy oil3.0 Limestone 1.307 Dicalcium phosphate 1.735 Salt (NaCl) 0.331 DLMethionine 0.186 Vitamin premix 0.25 Choline chloride 60% 0.05 CopperSulfate 0.05

Enzymes

Treat- Enzyme mG/L P val ment Enzyme 1 Enzyme 2 3 AGP T Test A — — —268.1 B 1,3-β-galactanase 1,4-β-mannanase — 281.4 (46,495 IU/ton)(85,312 IU/ton) C 1,3-β-galactanase 1,4-β-mannanase — 266.6 (9,8911IU/ton) (181,488 IU/ton) D 1,4-β-galactanase 1,4-β-mannanase — 261.7(81,046 IU/ton) (174,889 IU/ton) E 1,4-β-galactanase 1,4-β-mannanase —261.7 (114,418 IU/ton) (246,902 IU/ton) F xylanase 1,4-β-mannanase —257.8 (95,225 IU/ton) (381,142 IU/ton) G chitinase 1,4-β-mannanase —263.8 (5,218 IU/ton) (27,016 IU/ton) H chitinase 1,4-β-mannanase — 220.20.040 (5,218 IU/ton) (205,807 IU/ton) I 1,3-β-glucanase 1,4-β-mannanase— 236.9 0.125 (127,042 IU/ton) (181,488 IU/ton) J xylanase1,4-β-mannanase esterase 234.5 0.083 (126,758 IU/ton) (362,976 IU/ton)

The “esterase” in Treatment J is an uncharacterized enzyme from a B.lentus gene in the same operon with xylanase. The substrate for thisenzyme was not identified. Its assignment as an “esterase” is based onthe similarity of the DNA sequence of this gene to other known esterasegenes. The esterase activity was not determined, but would be similar tothe xylanase level if the two proteins have similar specific activities.

The 1,4-β-galactanase and 1,3-galactanase were measured using a reducingsugar assay with pectin substrate.

At 14 days, blood serum was collected from all birds and samples wereanalyzed for α1-acid glycoprotein (AGP) content as described above. Theaverage level of α-1-acid glycoprotein from each treatment group isshown in the table above.

As reflected in the table, relative to Treatment A (no enzyme),Treatments H, I and J resulted in reduction of serum AGP.

Treatment H (chitinase plus 1,4-β-mannanase) yielded significant resultsafter only two weeks of growth. Although the amounts of enzymes were atlevels that did not show a response in other Treatments (compare toTreatment G with a comparable amount of chitinase and Treatments E and Fwith a comparable amounts of 1,4-β-mannanase), the combination ofchitinase and 1,4-β-mannanase resulted in significant AGP reduction,that could not be predicted from the results obtained when only oneenzyme was used.

Treatment I (1,3-β-glucanase and 1,4-β-mannanase) yielded notableresults, although not clearly statistically significant in thisexperiment (P value of 0.125). In other tests of longer duration,treatment with 1,3-β-glucanase and 1,4-β-mannanase did have asignificant effect on AGP level.

Comparing Treatment J (xylanase, 1,4-β-mannanase, esterase; P=0.083 vsTreatment A control) to Treatment F (xylanase+1,4-β-mannanase, without“esterase”) reveals that Treatment J yielded a notable effect, whereTreatment F did not show AGP reduction.

Example 12

This example tests the hypothesis that altering a feed composition toinclude ingredients that stimulate the innate immune system willincrease serum APP levels.

A 21-day chicken broiler test was performed using a basal corn-soybeandiet with a soy-oil high-energy diet as control. To obtain test diets,the control diet was modified to contain practical materials suspectedto have immune stimulatory components while maintaining the sameapproximate equivalent nutritional value. The test diets included thefollowing variations:

Corn/soy/soy oil controlAV blend oil (animal vegetable blend oil) inclusion;soy lecithin inclusion;poultry meal inclusion;DDGS (distillers grains and solubles by-product from ethanolmanufacture) inclusionat 5% with soy hull;DDGS inclusion at 15% w/o soy hull.

The diets are described in more detail in the tables below.

The AV blend and soy lecithin are expected to contain phospholipidscomprising the innate immune system stimulator phosphatidlyserine. Thepoultry meal may contain phosphatidlyserine, hyaluronan, and variousmicrobial stimulators derived from the offal or secondary microbialgrowth that could occur before processing. The DDGS is expected tocontain abundant yeast residue, including cell walls comprisingα-mannan, 1,3-β-glucan and chitin, as well as potentially stimulatingnon-fermented carbohydrate polymers from the original fermentationsubstrate.

Diet Compositions

Percent Composition Component Diet #1 Diet #2 Diet #3 Diet #4 Diet #5Diet 6 Corn 7.35% CP 56.9707 56.2985 56.2985 59.878 47.7939 49.3523 Soymeal 48.5% 36.4392 36.5403 36.5403 29.048 29.0293 29.0284 CP Soy oil2.5279 0 0 1.7221 3.1922 2.6831 AV blend 0 3.0975 0 0 0 0 Soy lecithin 00 3.0975 0 0 0 Poultry BPM^(a) 65% 0 0 0 5.0 0 0 Soy hulls^(d) 0 0 01.0559 1.0586 0 DDGS^(b) 0 0 0 0 15 15 Limestone 1.3129 1.3118 1.31181.2224 1.4158 1.4293 Dicalcium phosphate 1.7527 1.7544 1.7544 1.7661.5615 1.5576 Salt 0.3312 0.3315 0.3315 0.2313 0.1416 0.1407DL-methionine 0.2404 0.241 0.241 0.2276 0.2419 0.2392 L-lysine HCl 0 0 00.0131 0.1402 0.1402 Vitamin premix 0.25 0.25 0.25 0.25 0.25 0.25 0.25%Mineral PMX 0.075 0.075 0.075 0.075 0.075 0.075 0.075% Choline chloride0.05 0.05 0.05 0.05 0.05 0.05 60% Copper sulfate 0.05 0.05 0.05 0.050.05 0.05 ^(a)poultry BPM (by product meal); ^(b)DDGS (distiller drygrain and solubles); ^(c)AV blend (animal vegetable blend oil);^(d)addition of soy hulls to the poultry meal diet equalizes the mannancontent to compensate for reduced soy meal

For each diet, chicken broilers (Cobb x Cobb) were grown in threePetersime battery cages with eight birds per cage (0.631 sq. ft. perbird). After 21 days, the serum AGP levels of each bird was analyzed asdescribed in previous examples.

The modified diets showed clear evidence of innate immune systemstimulation based on significant increases in serum AGP at 21 days, asshown in the following table. The data also underscores theopportunities to reduce immune stress caused by diet components inaccordance with the invention, such as by the use of compositionscomprising enzymes that degrade immune stress inducing ingredients.

Diet Addition Mg/L AGP T Test (P value vs. 1) 1 Control 221.9 2 AV blend301.2 0.01882 3 Lecithin 309.0 0.01052 4 Poultry Meal 265.8 0.08613 5DDGS w/hull 307.6 0.00002 6 DDGS 386.7 0.00275

Example 13

This example demonstrates the efficacy of compositions comprising1,3-β-glucanase in reducing immune stress associated with 1,3-β-glucan,which is present in feedstuffs and, by virtue of its association withfungal cell walls, is a molecular pattern apparently recognizeduniversally by the innate immune system of animals. The results showthat, like 1,4-β-mannanase, 1,3-β-glucanase reduces serum levels of APPand improves animal growth performance.

Chicken broilers (Cobb x Cobb) were grown from day 1 to 21 on thetypical low fat corn/soybean meal diet shown in the table below. In twocases, the diets were supplemented by uniformly spraying liquid enzymeconcentrate solutions prepared from B. lentus fermentations to applyeither 400,000 IU/ton 1,4-β-mannanase or 264,000 IU/ton 1,3-β-glucanase.(In this case, a ton represents 2000 lbs. or 907.4 kg.)

Diet Composition

Component Diet % Corn 7.35% CP 59.5757 Soy meal 48.5% CP 36.0474 Soy oil0.321 Limestone 1.3175 Dicalcium phosphate 1.7461 Salt 0.3294DL-methionine 0.2378 Vitamin premix 0.25% 0.25 Mineral PMX 0.075% 0.075Choline chloride 60% 0.05 Copper sulfate 0.05

For each diet type, birds were grown in three Petersime® battery cageswith eight birds per cage (0.631 sq. ft. per bird). After 21 days, serumAGP levels of each bird was analyzed as described in previous examples.Bird weights and feed consumed were determined utilizing standardprocedures and feed conversion was calculated. The results are shown inthe following table

WAFC (weight adjusted feed conversion) is calculated as follows:

WAFC=FC−2.204*((W−Wa)/3)

whereFC=weight of feed consumed/weight gainedWa=average weight of all birds in the trialW=average live weight gain per cage

T Test mg/L P vs. Treatment AGP contr. WAFC P value Control 255.4 1.47 a(no enzyme) 1,4-β-mannanase 184.1 0.011 1.39 ab 1,3-β-glucanase 157.70.001 1.26 c

Both 1,4-β-mannanase and 1,3-β-glucanase reduced the serum levels ofα1-acid glycoprotein (AGP). Both enzyme treatments reduced the weightadjusted feed conversion, and the reduction in the 1,3-β-glucanase fedgroup was statistically significant.

Example 14

A chicken broiler trial was conducted in Petersine® battery cages withthe feed and methods described in Example 13 above, except withdifferent enzyme treatments, as summarized in the table below.

Lyticase, a crude 1,3-β-glucanase product obtained by fermentation ofArthrobacter luteus, was obtained from the Sigma Chemical Company, St.Louis Mo. Lyticase activity was determined by the reducing sugar methoddescribed below and 60 MU/ton (equivalent to 240,000 IU/ton) wasapplied. According to the manufacturer, this product also contains otheractivities, including chitinase activity, that was not measured.

Treatment Enzyme (s) Dose (MU/ton) AGP (mg/L) 1 none 0 215.5 21,3-β-glucanase 3 213.7 3 1,3-β-glucanase 15 199.4 4 1,3-β-glucanase 30185.5 5 1,3-β-glucanase 60 201.0 6 1,3-β-glucanase 60 189.21,4-β-mannanase 100 7 1,3-β-glucanase 75 194.9 8 1,3-β-glucanase 90180.7 9 Lyticase 60 165.2 10 Xyloglucanase 100 162.2 (1 MU = 4000 IU)

Increased levels of 1,3-β-glucanase resulted in an increased effect onAGP level (e.g., a dose response) up to about 30 MU/ton (120,000IU/ton). Providing this type of animal feed with about 30 MU/ton(120,000 IU/ton) 1,3-β-glucanase is expected to reduce immune stress, asreflected in a reduced level of serum AGP and/or improved animal growthperformance.

The results also show that xyloglucanase was effective at reducing serumAGP levels. Xyloglucanase (EC 3.2.1.151) is a 1,4-β-glucanase withspecificity for xyloglucan, a structural polymer in plants.

With the exception of the Lyticase, all of the enzymes used in thisexample were produced by B. lentus. Lyticase is produced by A. luteuswhich has been reclassified as Cellulosimicrobium cellulans.Fermentation of A. luteus has been shown to produce multiple forms of1,3-β-glucanase. See, e.g., (Ferrer, P. Microb Cell Factories 5:10,2006, published online 2006 March 17. doi: 10.1186/1475-2859-5-10). Theresults above show that Lyticase reduced the chicken serum AGP at leastas well as the B. lentus 1,3-β-glucanase preparation, indicating thatthe source of the enzyme is not important. That is, enzymes from anysource can be used in accordance with the invention. It also is possiblethat chitinase (reported by Sigma to be present in Lyticase) may haveimproved the performance of the Lyticase treatment.

Example 15

The following assays can be used to assess enzyme activity

(I) Xyloglucanase

Xyloglucanase activity can be assayed using the following protocol:

DNS reagent:

10 g/L NaOH, 2 g/L phenol, 10 g/L dinitrosalicylic acid, 1200 g/Lpotassium sodium tartrate tetrahydrate is prepared daily. Immediatelybefore use, 0.5 g/L anhydrous sodium sulfite is added.

Standard Solutions and Standard Curve:

A series of D-(+)-mannose standard solutions dissolved in water in theconcentration range of 0.1 to 0.5 g/Liter are prepared. 0.6 mL of eachmannose standard (in duplicate or triplicate) is added to 1.5 mL DNSworking solution in 13×100 mm glass tubes. A sample with a 0.6 mLaliquot of water can be used as a reagent blank to zero thespectrophotometer. The solutions are heated in a boiling water bath for5 minutes, cooled to ambient temperature and absorbance is read at 550nm. The expected result is a linear dose response between 0.20 and 1.2O.D. units. The slope of the standard curve (O.D 550/g/L mannose) iscalculated from the linear portion of the curve only. With this slope,the value of the g/L of reducing sugar is determined in the enzymereactions.

Xyloglucan Substrate:

Xyloglucan (Tamarind) is obtained from Megazyme International IrelandLtd., Bray, Co., Ireland is dissolved at 5 g/L in 50 mM Tris buffer, pH7.5 with 0.05% glucose.

Reaction Conditions:

0.25 mL of 5 g/L xyloglucan substrate is used with a 0.05 mL enzymedilution in 50 mM Tris buffer, and the reaction mixture is incubated at40° C. 0.75 mL DNS reagent is added to stop the reaction, and thestopped reaction mixture is heated in boiling water bath for fiveminutes and then cooled prior to reading absorbance at 550 nm. A zerotime point with enzyme solution is used to determine the backgroundlevel.

Calculation:

A ChemGen xyloglucanase MU is defined as the ability to produce 0.72grams of reducing sugar per minute (using pure mannose, a reducingsugar, as standard). One ChemGen MU is equivalent to 4000 IU. In otherwords, one CG U is equivalent to 250 IU (IU=1.0μ mole/minute).

(II) B-1,3-Glucanase

B-1,3-glucanase activity can be assayed using the following protocol:

This assay uses the same DNS reagent, standard solutions, standardcurve, and enzyme unit calculation and dilution amount as describedabove for the xyloglucanase assay. The buffer used is a 50 mM MOPS(4-Morpholinepropanesulfonic Acid, FW=209.26) buffer at pH 6.5.

CM Pachyman Substrate:

Carboxymethyl Pachyman (CM Pachyman, CMP) is obtained from MegazymeInternational Ireland Ltd., Bray, Co., Ireland. CMP substrate isprepared at 5 g/L by slowly adding CMP into a fast stirring 50 mM MOPSbuffer solution (pH 6.5) at about 90° C. The enzyme powder iswell-dispersed, and the vessel is covered or sealed tightly, while thesuspension is heated slowly to boiling and simmered for 30 minutes withstirring on a heated-stir plate, to obtain a well-hydrated gel with nosmall clumps of non-hydrated gel visible in the solution. The solutionis cooled to room temperature, stored at 4° C. when not in use, andmixed well prior to use after storage.

Reaction Conditions:

0.25 mL of 5 gL CM Pachyman substrate is used with 0.05 mL enzymedilution in MOPS buffer and the reaction mixture is incubated at 40° C.for various times, up to 45 minutes. 0.75 mL DNS reagent is added tostop the reaction. The stopped reaction mixture is heated in boilingwater bath for five minutes and then cooled prior to reading absorbanceat 550 nm. A zero time point with enzyme solution is used to determinethe background level.

(III) Chitinase

Chitinase activity can be determined using the fluorogenic chitinsubstrate described in Thompson et al., Appl. Environ. Microbial. 67:4001-008 (2001),4-methylumbelliferyl-beta-O—N,N′,N″,N′″-tetraacetylchitotetraoside. Thesubstrate is dissolved in DMSO at 2.5 mM.

In an exemplary assay, 20 μL, chitin substrate (2.5 mM) is used with 150μL, tris (20.0 mM, pH 7.5). The substrate mixture is placed in a black98 well microtiter plate and pre-heated to 37° C. for 10 minutes.Multiple replicas of reactions are started by addition of 30 μL, dilutedenzyme and incubation is continued at 37° C. Individual reactions arestopped at 2, 4, 6, 8 and 10 minutes with 50 μL 3 MNa₂CaO₃. Fluorescenceis read in a microtiter plate reader (Fluoroscan II) using excitation355 nm band pass filter and emission 460 nm band pass filterwavelengths. The enzyme is diluted such that the 4-methylumbelliferoneis produced at a linear rate for the term of the reaction and within therange of a standard curve produced under conditions identical to theenzyme assay but without enzyme and substrate present. The release ofone micromole of 4-methylumbelliferone per minute is defined as one IU.A standard curve is made with several concentrations between zero and1×10⁻⁴ micromole 4-methylumbelliferone in 200 μL, reaction buffersolution followed by addition of 50 μL 3 MNa₂CaO₃.

While the invention has been described and exemplified in sufficientdetail for those skilled in this art to make and use it, variousalternatives, modifications, and improvements should be apparent withoutdeparting from the spirit and scope of the invention. The examplesprovided herein are representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of theinvention. Modifications therein and other uses will occur to thoseskilled in the art. These modifications are encompassed within thespirit of the invention and are defined by the scope of the claims.

It will be readily apparent to a person skilled in the art thai varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those of ordinary skill in the art to whichthe invention pertains. All patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

1: A composition suitable for oral administration to an animalcomprising an immune stress-reducing enzyme in an orally acceptablecarrier, wherein said composition is selected from the group consistingof: (i) an animal feed comprising an amount of said enzyme effective todecrease the level of positive acute phase protein in said animal,increase the level of negative acute phase protein in said animal,and/or improve animal growth performance; (ii) a liquid compositionother than an animal feed comprising at least 40,000 IU enzyme/L; and(iii) a solid composition other than an animal feed comprising at least40,000 IU enzyme/kg, wherein: (a) said enzyme is other than aβ-mannanase-type hemicellulase or phospholipase, and (b) if said enzymecomprises 1,3-β-glucanase, said composition is selected from the groupconsisting of (i) an animal feed comprising at least 20 IU1,3-β-glucanase/kg feed; (ii) a liquid composition other than an animalfeed comprising at least 155,000 IU 1,3-β-glucanase/L and (iii) a solidcomposition other than an animal feed comprising at least 300,000 IU1,3-β-glucanase/kg. 2-4. (canceled) 5: The composition of claim 1,wherein the composition is an animal feed that comprises an ingredientthat induces an immune response in the animal and wherein the enzymecomprises an enzyme that degrades said ingredient. 6: The composition ofclaim 5, wherein said ingredient is an antigen displayed by a pathogenicmicroorganism. 7: The composition of claim 1, wherein the enzymecomprises an enzyme selected from the group consisting of β-glucosidase,xyloglucanase, DNAases, non-specific nucleases, RNAse L, dsRNA specificadenosine deaminase, CG specific restriction endonuclease, N-glycanases,endo enzymes, PNGases, carbohydrases, α-1,2-fucosidase,α-1,3-1,4-fucosidase, α-1,6-mannosidase, α-1,2-mannosidase,α-1,3-mannosidase, β-1,4-galactosidase, endo-β-N acetylglucosaminidase F(endo F), peptide-N—(N-acetyl-beta-glucosaminyl)asparagine amidase F(PNGase F), PNGase A, endo-β-N-acetylglucosarninidase H (endoH), endo D,endo C, α-N-acetylgalacosamidase, β-1,3-galactosidase,endo-N-acyl-neuraminidase (endo N), α-2,3-neuraminidase,α-2,6-neuraminidase, α-2,8-neuraminidase, β-N -acetylhexosarninidase,endo-β-N-galactosidase, endo-α-N-acetylglactosaminidase,endo-α-1,6-D-mannanase, arabinogalactanase, α-mannanase, α-mannosidase,sphingomyelinase, chitinase, chitin deacetylase, carbohydratedeacetylase, N-acetylglucosaminidase, phosphatidylserine decarboxylase,sulfatase, β-galactosidase, arabinanase, hyaluronidase,α-arabinofuranosidase, chondroitinase, glucocerebrosidase, methylesterase, ferulic acid esterase, furuloyl esterase, acetyl esterase, andcarbohydrate deacetylase. 8: The composition of claim 1, wherein theenzyme comprises 1,3-β-glucanase. 9: The composition of claim 8, whereinthe composition is selected from the group consisting of (i) an animalfeed comprising at least 30 IU 1,3-β-glucanase/kg feed; (ii) a liquidcomposition other than an animal feed comprising at least 230,000 IU1,3-β-glucanase/L and (iii) a solid composition other than an animalfeed comprising at least 450,000 IU 1,3-β-glucanase/kg. 10: Thecomposition of claim 1, wherein the enzyme comprises an enzyme selectedfrom the group consisting of chitinase, xyloglucanase and arabinanase.11: A composition suitable for oral administration to an animalcomprising two or more immune stress-reducing enzymes, wherein saidcomposition comprises at least one immune stress-reducing enzyme otherthan 1,4-β-mannanase and 1,3-β-glucanase, and wherein said compositionis selected from the group consisting of: (i) an animal feed comprisingan amount of said immune stress-reducing enzymes effective to decreasethe level of positive acute phase protein in said animal, increase thelevel of negative acute phase protein in said animal, and/or improveanimal growth performance; (ii) a liquid composition other than ananimal feed comprising at least one immune stress-reducing enzyme in anamount of at least 40,000 IU enzyme/L; and (iii) a solid compositionother than an animal feed comprising at least one immune stress-reducingenzyme in an amount of at least 40,000 IU enzyme/kg. 12: The compositionof claim 11, wherein said composition is an animal feed comprising atleast one immune stress-reducing enzyme in an amount of at least 20 IUenzyme/kg feed. 13: The composition of claim 11, wherein the compositionis a solid composition other than an animal feed comprising at least oneimmune stress-reducing enzyme in an amount of at least 80,000 IUenzyme/kg. 14: The composition of claim 11, wherein the composition is asolid composition other than an animal feed comprising at least 160,000IU enzyme/kg. 15: The composition according to claim 11, comprising atleast one of 1,4-β-mannanase and 1,3-β-glucanase. 16: The composition ofclaim 11, comprising at least one enzyme selected from the groupconsisting of β-glucosidase, xyloglucanase, DNAases, non-specificnucleases, RNAse L, dsRNA specific adenosine deaminase, CG specificrestriction endonuclease, N-glycanases, endo enzymes, PNGases,carbohydrases, α-1,2-fucosidase, α-1,3-1,4-fucosidase,α-1,6-mannosidase, α-1,2-mannosidase, α-1,3-mannosidase,β-1,4-galactosidase, endo-β-N acetylglucosaminidase F (endo F),peptide-N—(N-acetyl-beta-glucosaminyl)asparagine amidase F (PNGase F),PNGase A, endo-β-N-acetylglucosarninidase H (endoH), endo D, endo C,α-N-acetylgalacosamidase, β-1,3-galactosidase, endo-N-acyl-neuraminidase(endo N), α-2,3-neuraminidase, α-2,6-neuraminidase, α-2,8-neuraminidase,β-N -acetylhexosarninidase, endo-β-N-galactosidase,endo-α-N-acetylglactosaminidase, endo-α-1,6-D-mannanase,arabinogalactanase, α-mannanase, α-mannosidase, sphingomyelinase,chitinase, chitin deacetylase, carbohydrate deacetylase,N-acetylglucosaminidase, phosphatidylserine decarboxylase, sulfatase,β-galactosidase, arabinanase, hyaluronidase, α-arabinofuranosidase,chondroitinase, glucocerebrosidase, methyl esterase, ferulic acidesterase, furuloyl esterase, acetyl esterase, and carbohydratedeacetylase. 17: The composition according to claim 11, wherein thecomposition is selected from the group consisting of (i) a compositioncomprising 1,4-β-mannanase and chitanase; (ii) a composition comprising1,4-β-mannanase and xyloglucanase; (iii) a composition comprising1,4-β-mannanase and arabinanase; (iv) a composition comprising1,3-β-glucanase and chitanase; (v) a composition comprising1,3-β-glucanase and xyloglucanase; (vi) a composition comprising1,3-β-glucanase and arabinanase and (vii) a composition comprising1,4-β-mannanase, 1,3-β-glucanase and arabinanase. 18: A compositionsuitable for oral administration to an animal comprising 1,4-β-mannanaseand 1,3-β-glucanase, wherein said composition is selected from the groupconsisting of (i) an animal feed comprising 1,4-β-mannanase and at least20 IU 1,3-β-glucanase/kg feed, (ii) a liquid composition other than ananimal feed comprising 1,4-β-mannanase and at least 155,000 IU1,3-β-glucanase/L and (iii) a solid composition other than an animalfeed comprising 1,4-β-mannanase and at least 300,000 IU1,3-13-glucanase/kg. 19: The composition of claim 18, wherein thecomposition is selected from the group consisting of (i) an animal feedcomprising 1,4-β-mannanase and at least 30 IU 1,3-β-glucanase/kg feed;(ii) a liquid composition other than an animal feed comprising1,4-β-mannanase and at least 230,000 IU 1,3-β-glucanase/L and (iii) asolid composition other than an animal feed comprising 1,4-β-mannanaseand at least 450,000 IU 1,3-β-glucanase/kg. 20: The composition of claim18, further comprising one or more additional immune stress-reducingenzymes. 21: A method of improving animal growth performance and/orreducing immune stress in an animal, comprising orally administering tosaid animal a composition according to claim
 1. 22-26. (canceled) 27:The method of claim 21, wherein the composition comprises at least oneenzyme selected from the group consisting of β-glucosidase,xyloglucanase, DNAases, non-specific nucleases, RNAse L, dsRNA specificadenosine deaminase, CG specific restriction endonuclease, N-glycanases,endo enzymes, PNGases, carbohydrases, α-1,2-fucosidase,α-1,3-1,4-fucosidase, α-1,6-mannosidase, α-1,2-mannosidase,α-1,3-mannosidase, β-1,4-galactosidase, endo-β-N acetylglucosaminidase F(endo F), peptide-N—(N-acetyl-beta-glucosaminyl)asparagine amidase F(PNGase F), PNGase A, endo-β-N-acetylglucosarninidase H (endoH), endo D,endo C, α-N-acetylgalacosamidase, β-1,3-galactosidase,endo-N-acyl-neuraminidase (endo N), α-2,3-neuraminidase,α-2,6-neuraminidase, α-2,8-neuraminidase, β-N -acetylhexosarninidase,endo-β-N-galactosidase, endo-α-N-acetylglactosaminidase,endo-α-1,6-D-mannanase, arabinogalactanase, α-mannanase, α-mannosidase,sphingomyelinase, chitinase, chitin deacetylase, carbohydratedeacetylase, N-acetylglucosaminidase, phosphatidylserine decarboxylase,sulfatase, β-galactosidase, arabinanase, hyaluronidase,α-arabinofuranosidase, chondroitinase, glucocerebrosidase, methylesterase, ferulic acid esterase, furuloyl esterase, acetyl esterase, andcarbohydrate deacetylase.
 28. (canceled) 29: A method of preventing ortreating infection associated with a pathogenic microorganism thatdisplays an antigen, comprising orally administering to an animal inneed thereof a composition according to claim 1, wherein the compositioncomprises at least one immune stress-reducing enzyme that degrades saidantigen.
 30. (canceled)